Incidence and visual outcome of endophthalmitis associated with intraocular foreign bodies

Purpose To determine the risk factors and visual outcome of endophthalmitis associated with traumatic intraocular foreign body (IOFB) removal and its allied management.
Introduction
Intraocular foreign body (IOFB) caused ocular trauma is a significant and unique type of trauma that requires skillful investigation and an early intervention. Endophthalmitis is an uncommon but potentially catastrophic complication of penetrating ocular injury with retained intraocular foreign bodies (IOFB) [1]. Studies have reported incidence of endophthalmitis ranging from none to as high as 13.5% [1–5]. Management of endophthalmitis associated with retained IOFB is a challenging subject. With advancement in vitreous surgery, a large number of eyes with ocular trauma and IOFB are being saved. Visual prognosis, however, is affected by the complexity of the confluent factors surrounding the IOFB, which include the size, site, material, trajectory, reactivity of foreign body, inflammatory response, degree and type of tissue damage, length of time since injury and any associated endophthalmitis [2]. Limited information is available regarding ocular trauma and IOFB-associated endophthalmitis outside of the United States and other developed countries [6–10]. In particular, very limited information on this entity is available from the Middle East. The current study was undertaken to analyze and report our experience at a large tertiary eye care referral center in the Middle East regarding the management of eyes with IOFBs and associated endophthalmitis. We identify the risk factors for the development of clinical endophthalmitis and visual outcome of such eyes at a major referral center in the Middle East.
Methods
A detailed retrospective review was conducted of all patients who presented to King Khaled Eye Specialist Hospital, a JCIA (Joint Commission International Accreditation, USA) accredited tertiary eye care referral center in Riyadh, Saudi Arabia, from January 1983 to August 2004 with penetrating ocular trauma and retained intraocular foreign bodies (IOFB). Methods of IOFB extraction has varied during these years. For simplicity, the study period was divided into the first decade from 1983 to 1993 and the second decade from 1994 to 2004. For the past decade pars plana vitrectomy has been used more often for the posteriorly located IOFBs. Prior to removal of the foreign body (FB) all adhesions around the FB were released and it was freed from encapsulation where indicated. All IOFBs were removed by using IOFB forceps. Where necessary the sclerotomy was enlarged to facilitate easy removal of the FB. Endophotocoagulation was applied to the retina adjacent to the site of the IOFB. After removal of the FB, vitrectomy was utilized to remove any remains of FB capsule or fibrous tissue with the vitrectomy cutter. Only those eyes which showed any clinical evidence of endophthalmitis after trauma and retained IOFB were included in the study. Patients demographic studied included, age at presentation, sex, place of trauma, occupation, mode of injury and time between injury and repair. Other parameters included initial and final best corrected Snellen visual acuity, entry and location of IOFB and associated cataract, vitreous hemorrhage, retinal detachment, development of endophthalmitis, diagnostic studies performed, treatment rendered, type and size of IOFB. Complications such as cataract, retinal detachment and secondary procedures performed were also noted. Causes of visual loss such as corneal scarring, cataract, retinal detachment, retinal scars and loss of an eye were also investigated. In particular, all eyes with a retained IOFB and associated endophthalmitis were investigated and analyzed in detail.
Statistical analysis was carried out using SPSS version 10 (SPSS Inc., Chicago, IL, USA). We studied the association between the variables using the chi-square test because the data was categorical. Further multiple logistic regression analysis was conducted to predict the factors that were associated with favorable or poor visual outcome. A P < 0.05 was taken as a level of statistical significance.
Results
Among the 589 eyes of 565 patients (90.3% males; 9.7% females; 24 bilateral) with retained intraocular foreign bodies (IOFB) after trauma, 44 eyes (7.5%) had evidence of clinical endophthalmitis at the time of initial evaluation or subsequent to removal of the IOFB (Table 1). The setting of the injury resulting in IOFB varied, with the majority occurring at the place of employment in younger patients. The size of the IOFBs recovered ranged from 0.5 mm to 18 mm (average 4.2 mm). At presentation visual acuity (VA) of 20/200 or better was found in 8 eyes (18.1%), 20/400 to counting fingers in 6 (13.6%) and hand motion (HM) to light perception (LP) in 30 eyes (68.2%) (Table 2). Delay in treatment was mostly due to late patient referral after the trauma or lack of understanding on the part of the patient about the urgency in presentation to the primary care center. Endophthalmitis was recognized preoperatively in 32 eyes (72.7%) and post-operatively in 12 eyes (27.3%). In general, the size of the IOFB posed no significant risk of causing endophthalmitis except in cases where larger IOFB was associated with a significant trauma to the eye. The most important predictive factor of developing endophthalmitis was delayed repair of the globe and removal of IOFB. A delay in intervention of more than 24-hours was associated with a risk of clinical endophthalmitis (Fig. 1). In fact, only 11 eyes (25%) which developed endophthalmitis were repaired and IOFB removed within 24 hours after their trauma compared with 33 eyes (75%) that were repaired and IOFB removed more than 24 hours after trauma (range 2–42 days). In other words, a delay of more than 2 days in the repair of the traumatic globe and removal of IOFB was associated with a significant risk of endophthalmitis development (P < 0.05). The composition of IOFB had no significant effect on the development of clinical endophthalmitis. In particular, eyes with wood IOFB did not appear to be associated with increased risk of endophthalmitis compared to eyes with metallic IOFBs. The age of the patient had no bearing on the development of clinical endophthalmitis. However, when age and delayed repair of the trauma and removal of IOFB was correlated, an association with increased evidence of endophthalmitis was observed. The risk of endophthalmitis development and poor visual outcome was less common in eyes where the location of IOFB was in the anterior chamber or lens compared to the eyes having IOFB in the vitreous or retina. Among the 10 eyes where the final vision of no light perception (NLP) was recorded after the treatment of IOFB trauma and associated endophthalmitis, only 2 eyes had anterior location of foreign bodies compared with 8 eyes where IOFB were found in the posterior chamber, the difference being significant (P < 0.05). There were 24 cases of endophthalmitis during the first decade (1983–1993) and 20 cases of endophthalmitis during the second decade (1994–2004) of the study; the difference however was not statistically different.
Table 1Reported incidence of endophthalmitis with IOFBStudiesEyes with endophthalmitis (total eyes)PercentBrinton et al. [11]11 (103)10.7Khan et al. [12]10 (198)5.1Williams et al. [3]14 (105)13.3Behrens-Baumann and Praetorius [5]14 (297)4.7Thompson et al. [1]34 (492)6.9El-Asrar et al. [4]13 (96)13.5Present study (Chaudhry et al.)44 (589)7.5Table 2Presenting and final visual acuity of patients with IOFB and endophthalmitisVisual acuity (VA)PresentingFinal20/20–20/6031120/80–20/2005920/400–CF64HM–LP3010NLP010VA visual acuity, CF count finger, HM hand motion, LP light perception, NLP no light perceptionFig. 1Patient with endophthalmitis associated with retained intraocular foreign body
Positive cultures were obtained from 17 eyes (38.6%) with clinical signs of endophthalmitis (Fig. 1). In addition, 10 eyes showed evidence of microorganisms on the Gram stain but no growth was observed in cultures. Staphylococcus and Streptococcus species were most often recovered (9 eyes); the other species included Hemophilus, Bacillus, Pseudomonas, Eikenella, Corynebacterium, Propionebacterium acnea and Escherichea coli. None of the eyes with Bacillus, Pseudomonas or Corynebacterium attained any useful vision. All eyes suspected of endophthalmitis were administered intravitreal antibiotics and in some cases the treatment was repeated to obtain resolution of the signs. Most common intraocular antibiotics administered included vancomycin, ceftazidime and amikacin. Systemic preoperative antibiotics were administered in eyes with signs of endophthalmitis at approximately the same frequency as eyes without any signs of clinical endophthalmitis. However, systemic antibiotics were continued postoperatively for longer duration in eyes with signs of infectious endophthalmitis.
Thirty-one eyes (70%) underwent primary pars plana vitrectomy and 20 eyes (45%) had pars plana lensectomy at the time of IOFB removal and primary repair. Secondary procedures included scleral buckle around 8 globes, endolaser or external cryotherapy in 10 eyes and gas tamponade using SF6/C3F8 in 10 eyes for retinal breaks or retinal detachments. In addition, 11 eyes required cataract surgery, 12 eyes developed retinal detachment requiring additional treatment after the removal of IOFB and treatment of endophthalmitis. Vitreous hemorrhage was noted in 24 eyes that were associated with posteriorly located IOFBs but none among the eyes with IOFBs located in the anterior part of the eye. The final VA was 20/200 or better (defined as a good visual outcome) in 20 eyes (45%). Twenty-four eyes (55%) had final VA of less then 20/200 (defined as poor visual outcome). Twenty eyes (45%) had a final VA of hand motion or worse (Fig. 2). The causes of final vision of HM or worse in 20 eyes were phthisis (Fig. 3) or enucleation in 10 eyes, retinal detachment with proliferative vitroretinopathy in 5 eyes and corneal or macular scar in 5 eyes. Overall, 22 eyes (50%) that developed endophthalmitis due to trauma and retained IOFB had improvement in their VA. Ten eyes (22.7%) with endophthalmitis became NLP compared to 58 eyes (10.6%) from the larger group which became NLP but had no evidence of endophthalmitis, the difference being significant (P < 0.05). Among the 10 eyes with endophthalmitis where the final vision of NLP was recorded after the treatment of IOFBs and associated endophthalmitis, only 2 eyes had anterior location of FBs compared to 8 eyes where FBs were found in the posterior chamber. Among the patients with endophthalmitis, 4 eyes (12.9%) from the vitrectomy group (31 eyes) had final vision of NLP, while from the eyes without vitrectomy (11 eyes), 5 eyes (45.4%) had final vision of NLP, the difference between these two groups being significant (P < 0.05).
Fig. 2Patient with light perception vision after treatment of endophthalmitis and removal of intraocular foreign bodyFig. 3Patient with phthisical left eye and no light perception after treatment of endophthalmitis and removal of intraocular foreign body
Discussion
Ocular trauma associated with retained intraocular foreign bodies (IOFB) constitutes a significant proportion (18–40%) of all ocular injuries requiring surgical management [1, 2]. Despite advances in vitreoretinal and microsurgical techniques, the management of these injuries remains a challenge. The risk of endophthalmitis developing after penetrating ocular injury with retained IOFB is relatively low with the current techniques of surgical repair [1]. The incidence of infectious endophthalmitis after retaining an IOFB has been reported to be as low as 0% to as high as 13.5% [3–5, 13], the mean incidence being 6.8% (Table 1). The results from the present study are in agreement with the above studies of endophthalmitis associated with retained IOFB. A delay in management of more than 24 hours was found to increase the risk of infectious endophthalmitis in the present study. From a large study of the National Eye Trauma System Registry, 91% of the eyes in which infectious endophthalmitis developed had such evidence at the time of initial evaluation [1]. Delay in primary repair at our tertiary care eye hospital in the Middle East was either due to the patient presenting late or being referred to our facility later in the course of trauma and retained IOFB. Despite methods of improved transportation, early diagnosis and referral to properly trained eye care professionals, morbidity due to IOFB associated trauma has not decreased over the last decade.
The risk of an infectious endophthalmitis developing with IOFB has been reported to increase with age [1]; although, no such trend was observed in the present study. However, when age and delayed repair of the trauma and removal of IOFB was correlated, an association with increased evidence of endophthalmitis in our group of patients was observed. These results are in agreement with previous observations that older patients with delayed primary repair do appear to have an increased susceptibility to developing infectious endophthalmitis compared with younger patients [1].
Visual acuity (VA) appears to be significantly affected in the eyes with IOFB that subsequently developed endophthalmitis. From a previous large study of retained IOFB, Roper-Hall [14] reported 555 cases over a 19 year period, where there were no bilateral cases: in 60 eyes IOFB was not removed, size of IOFB was recorded in only 89 eyes and the enucleation rate was 20%. From a more recent report of 96 cases of posterior segment IOFBs studied over a 14 year period with 8.6 months of average follow-up, endophthalmitis was recorded in 13.5% of cases [4]. The overall rate of 7.5% cases with endophthalmitis in the current study are within the reported range of larger series [1, 3–5, 14, 12].
Several preoperative and operative factors have been found to have prognostic value in the final visual outcome of the traumatized eye with retained IOFB and endophthalmitis. Initial VA is an indicator of final VA and is a well recognized predictor of visual outcome in most of the previously published studies on IOFBs [4, 15]. In our study, poor initial VA of HM or light perception was significant in predicting a poor visual outcome (Table 2). The risk of endophthalmitis development and poor visual outcome appeared to be significantly increased when the IOFBs were recovered from the posterior chamber (vitreous or retina), compared to the IOFBs recovered from the anterior chamber. Among the 10 eyes with endophthalmitis where the final vision of NLP was recorded after the treatment of IOFBs and associated endophthalmitis, only 2 eyes had anterior location of foreign bodies compared to 8 eyes where foreign bodies were found in the posterior chamber.
In our study, positive cultures were obtained in slightly more then one-third of the eyes with clinical signs of endophthalmitis. In general, the culture is often positive in clinically diagnosed endophthalmitis secondary to trauma than due to intraocular surgery. However, considering that our hospital is a tertiary eye care referral center, most of these patients were on topical as well as systemic antibiotics prior to their referral and obtaining cultures at the time of IOFB removal. This view is corroborated by the observations that 10 additional patients who had evidence of microorganisms on Gram stain showed no growth in culture. The microorganisms isolated from the eyes in the present study were similar to those reported in previous studies on the development of infectious endophthalmitis after IOFB trauma [1, 4]. Although, from many eyes suspected of infectious endophthalmitis, no definite organism was recovered. The fact that these eyes showed clinical improvement after intravitreal injection of antibiotics supports the notion that an infectious process was involved. In a recent study, reasonable visual outcomes with immediate intravitreal injection of antibiotics and delayed vitrectomy in patients with clinical features of endophthalmitis and retained IOFB have been achieved [16].
The role of vitrectomy in the treatment of penetrating ocular trauma has been considered a major advance in the last two decades [17]. Early vitrectomy has helped in attaining useful functional vision in 25–51% of the eyes with posterior segment trauma [17, 13], and in some series it has helped in significant survival of eyes without improvement of final visual outcome [17]. In our hospital, during the last decade, pars plana vitrectomy had been used more frequently for the posteriorly located IOFBs. Although there were fewer cases of IOFB-associated endophthalmitis during the second decade of the study period, statistically we did not find a difference between the two decades. In our study of endophthalmitis, only 12.9% of the eyes in which vitrectomy had been performed initially did the vision later become NLP compared to 38.5% in which the vision became NLP in the eyes which had not undergone vitrectomy as an initial procedure at the time of IOFB removal and repair of ocular trauma. Vitrectomy removes the vitreous and blood clot scaffold that provides a framework to the visual localization of IOFB; it helps to find retinal breaks and detachments which may not be visible in the setting of vitreous hemorrhage. Our results corroborate previous reports of beneficial effects of early vitrectomy in the setting of ocular trauma associated with IOFBs [13]. Removal of lens was not necessary in all cases with corneal entry wound because the lens injury was localized without interfering with view for vitrectomy. Removal of posterior segment IOFB by pars plana vitrectomy has been advocated because it provides direct viewing and controlled removal of the IOFB [3, 18]. With the advent of advanced instrumentation and viewing systems used in vitrectomy, most vitrectomies in our hospital were performed in the second decade compared to the first.
The size of IOFB has been found to be a significant predictive factor of poor visual outcome in the previous studies of IOFB removal [18]. A large IOFB is more likely to inflict severe damage at the time of entry because of its higher kinetic energy, leading to a poor visual prognosis [18]. However, when considering similar sized IOFBs, no particular association between the visual outcome and the size of IOFB in eyes which developed endophthalmitis was found in our study.
A preoperative retinal detachment may be present in 5–21% of the eyes with IOFB and has been reported to be an important risk factor for poor visual outcome [3, 19]. The timing of surgery in these eyes for the removal of IOFBs has been found to be an important prognostic factor for better visual outcome. Without delay, removal of IOFB has been found to reduce the chances of endophthalmitis [15, 19, 20]. In the absence of endophthalmitis, many studies have found no significant difference on the visual outcome when IOFB removal was delayed for several weeks [3, 18, 21]. Ahmadieh et al. reported a delay in IOFB removal of more then 4 weeks in 85% of their patients because a majority of their patients had war related IOFBs [22]. Lack of suspicion of IOFBs by primary care physicians has led to the delay in the treatment of up to 44% of patients resulting in a delay of up to 3 years in the diagnosis, treatment, or both, of IOFB [18, 21].
In conclusion, the data from this retrospective study reiterate the importance of prompt recognition of retained IOFBs and early repair of related injuries to prevent endophthalmitis and associated complications of visual loss. Final vision appears to be considerably determined by the presenting VA and severity of injury. Eyes with IOFBs in the anterior segment appear to have better prognosis compared to eyes having IOFBs in the posterior segment. Early vitrectomy at the time of IOFB removal may be beneficial for overall visual outcome of the operated eyes. Owing to the retrospective nature of our study and most of the reported studies and the various types of injuries in different settings, results from these studies are difficult to compare. It is hoped that with the advent of modern instruments such as wide-angle viewing systems and high-speed cutters, the chances of complications such as retinal detachment may be greatly reduced. A multi-center prospective study may be required to address some of the confounding factors in the management of IOFB and associated endophthalmitis.

endophthalmitis
foreign bodies
risk factors
trauma
visual acuity


The Notch-1 intracellular domain is found in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary cortical neurons

Notch signalling affects most aspects of development, not least the determination of neural stem cell fate. Here, we describe the presence of the Notch-1 intracellular domain (N1ICD) in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary rat cortical neurons as well as several other mammalian cell lines. We also demonstrate that these N1ICD-positive sub-nuclear bodies are distinct from premyelocytic leukaemia (PML) and SC35 bodies. Furthermore, using Notch deletion constructs we determined that a region C-terminal of amino acid 2094 is involved in targeting the N1ICD into sub-nuclear bodies. These findings have ramifications for nuclear architecture and gene transcription.
Notch signalling regulates many aspects of invertebrate and vertebrate development ranging from somite formation [7] to epithelial–mesenchymal interactions [20] and the determination of neural stem cell fate [23]. Notch signalling is also active in adult tissues including the brain where it is involved in synaptic plasticity and memory [3,19]. There are four mammalian Notch receptors (Notch 1–4), which undergo a series of sequential proteolytic cleavages. The first cleavage (S1) occurs in the Golgi by a furin-like convertase generating two fragments that remain non-covalently attached to form the mature Notch receptor at the cell surface. Ligands of the Jagged and Delta families, expressed on neighbouring cells, bind to the Notch receptor leading to the second cleavage (S2) by tumor necrosis factor alpha converting enzyme (TACE), which results in the shedding of the extracellular domain. The second cleavage is followed by a constitutive cleavage (S3) within the transmembrane domain of Notch by γ-secretase. This cleavage releases the Notch intracellular domain (NICD), which migrates to the nucleus [20,14,15] where it interacts with the C-promoter binding factor-1 (CBF-1), the mammalian homologue of Suppressor of hairless (Drosophila) and Lag-1 (C. elegans), also known as RBP-Jκ [16]. In the absence of NICD, CBF-1 forms a complex with repressor proteins, resulting in the suppression of gene transcription. Interaction with NICD displaces the repressor proteins and allows the entry of transcriptional activators such as p300/CBP and Mastermind, which convert the CBF-1 repressor complex into an activator of gene transcription [8]. Notch/CSL target genes include the hairy and enhancer of split (HES) family and the HES related proteins (HERP/Hey) that encode basic helix-loop-helix transcription factors, which suppress differentiation by antagonising the expression of down-stream lineage-specifying genes. In this report, we demonstrate the presence of the N1ICD in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary rat cortical neurons.
To undertake this study the following cell lines, antibodies and plasmid constructs were used: SH-SY5Y neuroblastoma cells and chinese hamster ovary cells (CHO) were obtained from ECACC (UK) and human embryonic kidney 293a cells (HEK293a; an adherent clone of HEK293 cells) were purchased from Quantum Biotechnologies (Canada). Mouse monoclonal anti-splicing factor SC-35 antibody and rabbit polyclonal anti-FLAG tag antibody were from Sigma (UK). Mouse monoclonal anti-myc antibody was from Cell Signalling Technology (UK). Mouse monoclonal anti-promyelocytic leukaemia protein antibody, goat anti-Notch-1, goat anti-mouse IgG and donkey anti-goat IgG were from Santa Cruz Biotechnology (USA). Alexa Fluor 488 and 594 goat anti-mouse and donkey anti-goat IgG conjugates were from Molecular Probes (UK). C-terminally tagged myc-His human N1ICD, ΔTAD-N1ICD (lacking the transactivation domain TAD; the OPA domain and the proline (P), glutamate (E), serine (S), threonine (T) rich sequence—PEST) and ΔRA-N1ICD (lacking the RAM domain and the Ankyrin repeats) were gifts from Dr. Tom Kadesch (University of Pennsylvania, USA) [13]. Enhanced green fluorescent protein (EGFP) C-terminally tagged human N1ICD (N1ICD-EGFP) was from Dr. Allan Levey (Emory University School of Medicine, USA) [11]. The Notch-CBF-1 reporter, 4xwt-CBF-Luc, which contains four tandem repeats of the consensus CBF-1 DNA binding sequence, GTGGGAA and N-terminally tagged FLAG CBF-1 were generous gifts from Dr. Diane Hayward (Johns Hopkins University School of Medicine, USA) [5].
Cells were maintained in 5% CO2 in air in a humidified incubator at 37 °C. All culture media and supplements were from Invitrogen, UK, unless otherwise stated. SH-SY5Y neuroblastomas were grown in a 50/50 mix of F12/Eagle's minimal essential medium (EMEM) supplemented with 15% (v/v) fetal bovine serum (FBS; Autogen Bioclear, UK), 2 mM l-glutamine, non-essential amino acids (Sigma), 100 IU penicillin and 100 mg/ml streptomycin. HEK293a were cultured in low glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) FBS, 2 mM l-glutamine, 100 IU penicillin and 100 mg/ml streptomycin. CHO cells were grown in F12 media supplemented as described above for HEK293a cells. Primary cortical neurons were prepared and cultured in Neurobasal media plus B27 supplement as previously described from E19 rat embryos [17].
Cells on coverslips were transfected with 500 ng of N1ICD-EGFP or 500 ng of myc-His N1ICD using FuGene 6 according to the manufacturer's instructions. Cells were fixed in ice-cold methanol 24 h after transfection then stained according to standard protocols. In brief, cells were incubated with goat anti-Notch-1 antibody (1:100), mouse monoclonal anti-promyelocytic leukaemia protein antibody (1:100) or mouse monoclonal anti-splicing factor SC-35 antibody (1:250) before being incubated with the appropriate fluorescent secondary antibody (1:200). Nuclei were stained with Hoescht 33342. Immunofluorescence was visualized and captured using a Zeiss LSM510 meta confocal microscope. Images were processed using LSM5 image examiner software (Zeiss). All experiments were performed in triplicate. Figures shown are representative images from a single experiment.
For luciferase reporter gene assays SH-SY5Y neuroblastomas were transfected with 400 ng of CBF-Luc (firefly luciferase based reporter DNA) and 500 ng of N1ICD-EGFP or 500 ng of myc-His N1ICD using FuGene 6 according to the manufacturer's instructions (Roche, UK). To control for transfection efficiency 50 ng of phTK-Renilla luciferase (Promega, UK) was also included in transfections. Empty vector DNA was included where necessary to maintain constant DNA concentrations. Twenty-four hours post-transfection the cells were lysed and firefly and Renilla luciferase activities were sequentially measured using Dual-Glo reagents (Promega) in a Wallac Trilux 1450 Luminometer (Perkin-Elmer, UK). Values were normalized by dividing firefly values by the Renilla value from the same well. Data for each set of four replica transfections was averaged, the control in each set normalized to 1 and data presented as fold increases over control. All experiments were performed in triplicate.
Endogenous S3 cleaved, nuclear, NICDs are expressed at very low levels and are difficult to detect in cell culture experiments [9], therefore we expressed exogenous N1ICD-EGFP in SH-SY5Y neuroblastomas to model N1ICD in vitro. In these cells N1ICD-EGFP exhibited a diffuse nuclear staining pattern with intense sub-nuclear bodies (Fig. 1A and B). In contrast, expression of exogenous p53 or β-catenin resulted in diffuse nuclear staining (data not shown), providing evidence that the N1ICD-positive sub-nuclear bodies are specific. The sub-nuclear bodies were not attributable to the EGFP protein since SH-SY5Y cells transfected with the EGFP expression vector containing no insert did not display a speckled nuclear staining pattern (data not shown). Furthermore, a myc-His tagged form of N1ICD was found in sub-nuclear bodies in SH-SY5Y neuroblastoma cells (Fig. 1C) as demonstrated using an anti-Notch-1 antibody, providing additional evidence that the observed speckled staining pattern is specific and not due to the presence of EGFP. Control cultures that were not exposed to primary antibody did not exhibit any positively stained cells (data not shown). To ensure the presence of nuclear bodies was not an artefact of the fixation method used (ice-cold methanol), SH-SY5Y cells were transfected with both EGFP and myc-His tagged Notch constructs and fixed with either 3.7% formaldehyde or 4% paraformaldehyde. With both methods very similar nuclear bodies were seen with both constructs (data not shown). Of note, both N1ICD-EGFP and myc-His tagged N1ICD were functionally active as demonstrated using CBF-Luc (Fig. 1D) and we have previously shown that exogenous N1ICD expression promotes the inhibition of retinoic acid-induced differentiation in SH-SY5Y neuroblastomas [4].
N1ICD-positive sub-nuclear bodies were not cell-type specific. We observed identical sub-nuclear structures in primary rat cortical neurones (Fig. 2A), HEK293a cells (Fig. 2B) and in CHO cells (Fig. 2C) with N1ICD-EGFP and myc-His tagged N1ICD (data not shown). As the punctate structures were detected with both constructs in a variety of cell lines and in primary neurons they are unlikely to be an artefact of tagging or a result of cellular transformation. Furthermore, we found that exogenous CBF-1 exhibited a diffuse nuclear staining pattern in SH-SY5Y neuroblastomas (Fig. 2D), but in the presence of N1ICD CBF-1 re-located into sub-nuclear bodies (Fig. 2E), indicative of functional gene transcription within these nuclear domains.
Several sub-nuclear structures, similar to those shown here, have been previously described including premyelocytic leukaemia (PML) bodies and SC35 bodies. These foci are believed to be areas of high transcriptional activity [2] and pre-mRNA splicing, respectively [10]. We therefore transfected SH-SY5Y cells with N1ICD-EGFP and used antibodies to detect endogenous PML and SC35 bodies. Both PML (Fig. 3A, mid left panel) and SC35 reactive bodies (Fig. 3B, mid left panel) were detected in SH-SY5Y cells. However, in both cases these formations were distinct from the N1ICD-EGFP positive foci (Fig. 3A and B, far right panels). The PML and SC35 bodies were more consistent in size and typically smaller and more numerous than the formations in which N1ICD was detected.
Next we examined which region of N1ICD was involved in targeting the protein to sub-nuclear bodies by using two myc-tagged N1ICD deletion constructs, ΔTAD-N1ICD and ΔRA-N1ICD. ΔTAD-N1ICD runs from a.a. 1760 to 2093 and lacks the transactivation domain, the OPA domain and the proline (P), glutamate (E), serine (S), threonine (T) rich sequence (PEST). ΔRA-N1ICD extends from a.a. 2094 to 2556 and lacks the RAM domain and the Ankyrin repeats (Fig. 4A). ΔTAD-N1ICD was detected diffusely throughout the nucleus in SH-SY5Y neuroblastomas (Fig. 4B, left panel), while ΔRA-N1ICD was located in sub-nuclear bodies very similar in appearance to those seen with N1ICD (Fig. 4B, right panel). This indicates that a region C-terminal of a.a. 2094, lying within the TAD, the OPA domain or the PEST domain or an unidentified motif within the intervening regions, is involved in targeting N1ICD into sub-nuclear bodies.
In summary, we demonstrate that exogenous N1ICD is expressed in sub-nuclear bodies in SH-SY5Y neuroblastomas, primary rat cortical neurons as well as other mammalian cell lines and these sub-nuclear structures were distinct from SC35 or PML bodies. N1ICD foci were detectable as early as 5 h after transfection (cells were routinely fixed 24 h post-transfection in all other experiments described in this report), which suggests that the nuclear inclusions are not merely a consequence of protein aggregation with time (data not shown). The presence of the N1ICD in sub-nuclear bodies has previously been demonstrated when associated with the Epstein-Barr virus derived protein, RPMS [24] and the Mastermind family of Notch/CBF-1 co-activators [18,21,22]. Here, we report that the N1ICD appeared in foci independently of other exogenously expressed proteins. A number of endogenous Notch associated proteins, including CBF-1, have recently been observed in similar nuclear foci in primary hippocampal neurons [12], although the presence of NICDs was not examined. Interestingly, we found that exogenously expressed CBF-1 exhibited a diffuse nuclear staining pattern, however in the presence of exogenous N1ICD CBF-1 was found in sub-nuclear bodies. A number of previous reports [1,6] concerning Notch cytochemistry contain figures in which N1ICD appears to be in punctate formations, which with greater resolution could have been interpreted as distinct sub-nuclear bodies. It is feasible then that the N1ICD-positive bodies form part of a physiologically relevant, multimeric, protein complex involved in gene transcription. Moreover, the presence of such sub-nuclear bodies provides evidence that the nucleus contains defined domains and is not merely a homogenous pool of nucleic acids and proteins.

notch
sub-nuclear bodies
transcription
cbf-1
luciferase


Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3-5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.
Introduction
The roles of extracellular purine nucleosides and nucleotides as neurotransmitters and modulators are well documented [1]. Extracellular purines also exert trophic effects on cells; influencing growth, division [2, 3] differentiation, and even apoptosis [4–6]. Purines are released from cells under physiological conditions, acting as neurotransmitters and neuromodulators [7–9]. Trauma and other insults to the central nervous system are also potent stimuli that cause the release of purines [3], most of which are converted by ectoenzymes to adenosine and guanosine [3, 5]. After insults to cells, more guanine-based than adenine-based purines are released [10]. Furthermore, the extracellular concentration of guanosine remains elevated for prolonged periods in vitro [10] and for up to a week after central nervous system (CNS) injury [11], implying that extracellular guanosine may exert trophic effects in vivo.
Sprouting of neurites, which later become axons and dendrites, is an important change associated with neural development and differentiation [12, 13]. Neurite sprouting through regeneration or collateral sprouting also plays an important role in the functional recovery following injury to the central or peripheral nervous systems [14]. Amongst its trophic effects, guanosine stimulates neurite outgrowth and enhances NGF-dependent neurite outgrowth [3, 15].
PC12 cells serve as a useful model for studying cell signaling [16]. They respond to many growth factors, neurotrophins and hormones, which initiate multiple signaling pathways [17–20]. The specific cellular targets of these signaling pathways mediate the distinct responses of differentiation, proliferation and survival, all of which can be assessed [16]. The addition of NGF to PC12 cells causes a sustained activation of ERK, a mitogen-activated protein kinase, through activation of the TrkA receptor [16, 19]. This leads to the development of many phenotypic characteristics in PC12 cells, which are also associated with mature sympathetic neurons [13, 21].
Extracellular guanosine not only stimulates neurite outgrowth from primary cultures of rat hippocampal neurons [3], and PC12 cells [15] but also enhances the neuritogenic effects of NGF on PC12 cells [15, 22, 23]. Both in astrocytes [24] and in PC12 cells [22], guanosine increases intracellular adenosine 3′,5′-cyclic monophosphate (cAMP). Some of the neuritogenic effects of guanosine in PC12 cells appeared to be mediated through this mechanism [22]. However, guanosine also has cAMP-independent effects, synergistic with NGF, because its effects are not abolished by an adenylate cyclase inhibitor [22] and are synergistic with substances that increase intracellular cAMP [15].
The cAMP-independent component of the signal transduction mechanism through which guanosine synergistically enhances NGF-dependent neurite outgrowth is unknown. However, several lines of evidence indicate a role for guanosine 3′,5′-cyclic monophosphate (cGMP). First, extracellular guanosine increases intracellular cGMP in rat mesenteric artery [25]. Second, nitric oxide (NO) donors enhance NGF-dependent PC12 cell neurite outgrowth via a cGMP-dependent mechanism; and thirdly, the neuritogenic effects of the NO donors are abolished by guanylyl cyclase inhibitors and mimicked by cGMP analogs [26]. The mechanism by which cGMP enhances neurite outgrowth is currently unclear.
Nitric oxide synthases are a family of enzymes, which synthesize NO through the catalytic conversion of l-arginine to l-citrulline. The constitutively expressed forms are the endothelial (eNOS) or neuronal (nNOS), which are regulated by the cytosolic concentration of Ca2+ [27]. The inducible isoform (iNOS) is widely distributed, and becomes active only hours after an inducing event [28]. NO is involved in cell communication and signal transduction in many systems including the CNS [29–31]. NGF induced different isoforms of NOS in PC12 cells after 4 days in vitro [32, 33]. Peunova and Enikolopov [32] reported that the NO produced preceding the development of the differentiated phenotype is due predominantly to iNOS.
The diffusible gas carbon monoxide (CO) is a putative neurotransmitter [34]. Heme oxygenase (HO) synthesizes CO from the biologically active substrate biliverdin, which is rapidly reduced to bilirubin, and iron from intracellular heme [34]. CO can modulate activities attributed to cGMP in the nervous system [35–38]. It has been proposed that CO production is responsible for baseline cGMP levels in the hippocampus [36]. In cerebellar granule cell cultures, CO produced by HO affects intracellular cGMP concentrations by modulating the NO-soluble guanylate cyclase signaling system [39]. Conversely, NO, synthesized by NOS may induce the expression of HO-1 [40–42], indicating a close and reciprocal interaction between the NOS-NO and the HO-CO signaling systems [35, 36]. Based on these findings it has been proposed that a possible role for HO-1 is to counteract NO toxicity [43].
Together, these data led us to question whether guanosine enhanced NGF-dependent neurite outgrowth is through a mechanism involving cGMP, and if so, whether it was attributable to the stimulation of either NO or CO synthesis.
Materials and methods
Cell culture and treatments
Tissue culture supplies were from Life Technologies. All other supplies were obtained from Sigma RBI unless otherwise stated. 2.5S NGF was a generous gift from Dr. M. Coughlin, Department of Medicine, McMaster University, 6-(phenylamino)-5,8-quinolinedione (LY83583) was obtained from (Calbiochem), copper protoporphyrin from (Porphyrin Products), and zinc protoporphyrin IX from (Research Biochemical).
PC12 cells were maintained in either RPMI 1640 medium supplemented with 5% heat-inactivated (HI) fetal calf serum (FCS), 5% HI-horse serum (HS) and 1% antibiotic-antimycotic (Anti-Anti; 10,000 units of penicillin, 10,000 µg of streptomycin, 25 µg amphotericin B/ml in 0.85% saline) [15] or F-12K (Kaighn's Modification) medium supplemented with 15% HI-HS, 2.5% HI-FCS, and 1% Anti-Anti at 37 °C in a 5% CO2 environment.
Neurite outgrowth assay
To evaluate the effect of test compounds on neurite outgrowth in PC12 cells, they were added to cultures for 48 h as previously described [15]. Briefly, PC12 cells were plated onto poly-d,l-ornithine (PORN)-coated 24-well plates at a density of 2.5 × 104 cells/well. Cells were cultured in RPMI 1640 supplemented with 1.5% HI-HS, 1.5% HI-FCS and 1% Anti-Anti. Guanosine was dissolved in 10% sodium hydroxide (1N NaOH) and when added to the culture medium the final concentration was 0.01% sodium hydroxide. In experiments in which guanosine (300 µM) was added to each well first, followed within 15 min by the addition of 2.5S NGF (40 ng/ml). Methylene blue, LY83583, hemoglobin, Nω-nitro-l-arginine methyl ester (l-NAME), copper protoporphyrin-IX (CuPP) and zinc protoporphyrin-IX (ZnPP), were added 15 min prior to the addition of guanosine. All test substances remained in the medium throughout the 48-h test period. Cells were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO2. After 48 h, cells were fixed with 10% formalin in phosphate buffered saline (PBS), pH 7.4 for 10 min and stored at 4 °C in PBS (pH 7.4) containing sodium azide (0.1%, w/v) until counted (usually within 2 weeks). Total cell number was determined by counting two randomly selected areas in each well. The observer was blind to the treatments the cells had received. At least 50 cells per well were counted, and the number of cells bearing one or more neurites determined using a NIKON Diaphot microscope equipped with phase contrast optics. Neurites were defined as processes extending at least one cell body diameter from the cell with growth cones at their tips [44].
Western immunoblot analysis
The expression of HO-1, HO-2, iNOS, nNOS, and β-actin in PC12 cells was determined by Western immunoblot analysis. PC12 cells were seeded onto PORN-coated plates at a density of 1 × 106 cells/plate and maintained at 37° for 48 h prior to treatments. Cells were placed in a medium containing 3% HI-FCS and 3% HI-HS 12 h before the start of treatments. Cells were then treated with various agents for 0, 6, 12, 24, or 48 h. At the end of the treatment period cells were washed once with PBS and then harvested at 4 °C using a lysis buffer (25 µM Tris/HCl pH 7.4, 10 µM NaCl, 10 µM EDTA, 100 µl/10 ml Tween 20, 10 µM sodium pyrophosphate decahydrate, 10 mM sodium orthovanadate, 5 µg/ml leupeptin, 10 mM glycerophosphate). Cells were disrupted by sonication and aliquots (25 µl) were removed for the determination of protein concentrations using the bicinchoninic acid (BCA) protein assay (Pierce, Illinois, USA). Cells lysates were separated on 12% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (PALL, Michigan, USA). Membranes were incubated with a specific primary antibody overnight at 4 °C then were exposed to a secondary antibody for 1 h at room temperature. Positive control peptides for nNOS and iNOS (obtained from Cayman Chemical, Ann Arbor. MI) were used as electrophoresis standard. The following antibodies were used: monoclonal antibody to brain NOS (Sigma, dilution: 1:1,000), monoclonal antibody to the inducible NOS (Sigma, dilution: 1:1,000), anti-iNOS/bNOS, goat anti-mouse IgG-HRP (StressGen Biotechnologies, dilution 1:100,000), monoclonal antibody to β-actin (abCAM, dilution: 1:20,000), monoclonal anti-β-actin, goat anti-mouse IgG-HRP (Novus Biological, dilution 1:100,000), monoclonal antibody to HO-1 (StressGen Biotechnologies, dilution 1:2,000), anti-HO-1, goat anti-mouse IgG-HRP (StressGen Biotechnologies, dilution 1:200,000), rabbit polyclonal antibody to HO-2/HO-1 (StressGen Biotechnologies, dilution 1:1,000), anti HO-2/HO-1, goat anti-rabbit IgG-HRP (StressGen Biotechnologies, dilution 1:200,000). Immunocomplexes were then visualized using a chemiluminescence substrate (Sigma-Aldrich). Immunoblots were quantified by densitometric analyses, using the Northern Eclipse program (EPIX). All bands on the immunoblots were normalized to their corresponding β-actin bands prior to statistical analysis.
Determination of cyclic GMP
PC12 cells were seeded onto PORN-coated 12-well plates at a density of 5 × 105 cells/well and maintained at 37 °C for 48 h prior to treatments. Cells were placed in a medium containing 3% HI-FCS and 3% HI-HS 12 h before the start of treatments. Cells were then treated with various agents for different times (0, 6, 12, 24, or 48 h). At the end of the treatment period cGMP was extracted and analyzed using a cGMP enzyme immunoassay (EIA) kit (Amersham Biosciences). The assay is based on a competition between unlabeled cGMP in the sample or (standard) and a fixed quantity of peroxidase-labeled cGMP for a limited number of binding sites on a cGMP specific antibody. With fixed amounts of antibody and cGMP-peroxidase conjugate, the amount of bound cGMP-peroxidase conjugate is inversely proportional to the concentration of unlabeled cGMP.
Statistical analysis
Statistical analysis was carried out using a two-way ANCOVA, when applicable, or a two-way ANOVA followed by Fischer's LSD test for multiple comparisons.
Results
Guanosine enhances NGF-dependent neurite outgrowth via activation of soluble guanylate cyclase
As we have reported previously, we have evaluated the effect of guanosine on NGF-dependent neurite outgrowth during the first 48 h, as guanosine most likely exerted its effects within the first 1–2 h after treatment [23]. Therefore, all experiments reported in this manuscript were performed during the first 48 h of PC12 cell treatment with various agents.
PC12 cells contain both soluble, and particulate isoforms of guanylate cyclase (GC) [26, 45]. To determine whether activation of GC was necessary for the neuritogenic effect of guanosine, inhibitors of GC were added for 48 h to PC12 cells treated with NGF plus guanosine. Methylene blue at concentrations ranging from 0.1 to 1 mM was added to PC12 cell cultures that contained NGF alone (40 ng/ml), or guanosine (300 mM) plus NGF (40 ng/ml). Cultures were then evaluated for neurite outgrowth. At the concentrations used in these experiments, methylene blue inhibits sGC but not particulate GC [46, 47]. Methylene blue had no effect on the outgrowth of neurites induced by NGF alone (Figure 1). However, in cultures treated with both guanosine and NGF, the addition of methylene blue reduced significantly (P < 0.01) the proportion of neurite-bearing cells in a concentration-dependent manner (Figure 1). LY83583 inhibits both, particulate GC and sGC [48]. When LY83583 (10 nM) was added to PC12 cell cultures, it inhibited neurite outgrowth elicited by guanosine plus NGF but had no effect on neurite outgrowth in cultures treated with NGF alone (data not shown). These data support the hypothesis that cGMP plays a role in enhancing the effect of guanosine on NGF-mediated neurite outgrowth. Moreover, since methylene blue at the concentrations used inhibits soluble but not particulate GC, these data imply that guanosine activates sGC.
Figure 1
Methylene blue attenuates guanosine-enhanced nerve growth factor-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum, 5% heat-inactivated horse serum and 1% antibiotic-antimycotic for 48 h. Cultures were then treated with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) and with increasing concentrations of methylene blue (0–1 µM). After 48 h, the total cell number and number of cells bearing one or more neurites were determined by counting two random areas in each well. The mean proportion of neurite-bearing cells in cultures treated with NGF was approximately 25%–35%. Because this value varied slightly between experiments, all experimental values are expressed relative to the NGF treated cultures, which was defined as 100%. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Cultures treated with guanosine plus NGF had a significantly (P < 0.01, two-way independent ANOVA) greater proportion of neurite-bearing cells than those treated with NGF alone. Methylene blue (0.1 to 1.0 µM) had no effect on the proportion of neurite-bearing cells in cultures treated with NGF alone, but at concentrations from 0.1 to 1.0 µM, it significantly (**P < 0.01, two-way independent ANOVA) reduced the proportion of neurite-bearing cells in cultures treated with guanosine plus NGF. Data represent the mean ± SEM of 12 determinations from two replicate experiments.
Guanosine may activate sGC either directly, or indirectly by stimulating the formation of NO, CO or hydroxyl radicals, which are all physiological activators of sGC [49]. To distinguish between these possibilities we added hemoglobin (100 nM) to cultures of PC12 cells. Extracellular hemoglobin directly scavenges NO and CO because these substances avidly bind to the heme moiety, thereby inhibiting their GC activating activity [46]. Hemoglobin reduced the synergistic effects of guanosine on NGF-mediated neurite outgrowth but had no effect on neurite outgrowth elicited by NGF alone (data not shown). Although these data imply that guanosine stimulates the synthesis of a sGC-activating factor, they do not show whether this factor is NO, CO or hydroxyl radical.
Inhibition of nitric oxide synthase (NOS) has no effect on guanosine-enhanced NGF-dependent neurite outgrowth
In the rat mesenteric artery, guanosine stimulates NO formation [25]. Therefore, we investigated if guanosine could also stimulate NO synthesis in PC12 cells. To examine whether NO was involved in the signal transduction pathway that mediated the enhancement of neurite outgrowth by guanosine through sGC, we inhibited the enzyme NOS, which catalyzes the conversion of l-arginine to l-citrulline plus NO. The l-arginine analog, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) [50] inhibits NOS competitively [25, 51]. In this experiment, we reduced the concentration of l-arginine in the culture medium from 1 mM to 80 µM, thus permitting more effective competition of the NOS inhibitor with l-arginine. Under these conditions, L-NAME inhibits NOS at 10 µM [25, 51]. When added to the culture medium of PC12 cells at 10 µM l-NAME did not inhibit NGF-dependent neurite outgrowth, and had no effect on the guanosine-mediated enhancement of NGF-dependent neurite outgrowth (Figure 2a). To confirm that NO was not involved in the signal transduction pathway that mediates the enhancement of neurite outgrowth by guanosine, we determined the expression of two subtypes of NOS isoenzymes: iNOS, the inducible isoform (Figure 2b), and nNOS, the constitutive isoform (Figure 2c). PC12 cultures were exposed to guanosine alone (300 µM), or NGF alone (40 ng/ml), or to the combination of guanosine plus NGF for 48 h, and NOS expression was determined by Western immunoblot analysis. We analyzed various protein concentrations (5–50 µg/ml) and determined β-actin expression on the same Western immunoblots. Using protein concentrations as high as 50 µg/ml neither nNOS nor iNOS expression could be detected in PC12 cells treated with guanosine, or NGF, or the combination of the two agents. These data indicate that since NOS is not expressed during the first 48 h of treatment, thus NO cannot mediate the effect of guanosine on neurite outgrowth.
Figure 2
(a) Inhibition of nitric oxide synthase has no effect on the proportion of neurite-bearing PC12 cells cultured for 48 h in the presence of NGF, or NGF plus guanosine. PC12 cells were cultured with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) as described in Figure 1. Cultures were treated with the general nitric oxide synthase inhibitor, l-NAME (0.1–10 mM) for 48 h and the number of cells bearing one or more neurites were determined as described in Figure 1. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Neurite outgrowth in cultures treated with guanosine plus NGF was significantly (P < 0.01, two-way independent ANOVA) greater than in cultures treated with NGF alone. l-NAME (0.1–10 µM) had no effect on the proportion of neurite bearing cells in cultures treated with NGF alone, or in combination with guanosine. Data represent the mean ± SEM of 12 determinations from two replicate experiments. (b) Inducible nitric oxide synthase is not expressed in PC12 cells cultured for 48 h in the presence of guanosine, or NGF, or guanosine plus NGF. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C). The expression of inducible nitric oxide synthase was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Recombinant inducible nitric oxide synthase protein (50 ng) was used as a positive control (P). Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test. Data presented are representative of at least three independent experiment. (c) Neuronal nitric oxide synthase is not expressed in PC12 cells cultured for 48 h in the presence of guanosine, or NGF, or guanosine plus NGF. PC12 cells were cultured as described in panel (b), and the expression of neuronal nitric oxide synthase was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Recombinant neuronal nitric oxide synthase protein (50 ng) was used as a positive control (P). Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test. Data presented are representative of at least three independent experiment.
Inhibition of heme oxygenase (HO) attenuates guanosine-enhanced NGF-dependent neurite outgrowth
Since NO apparently did not mediate the effects of guanosine, we examined the possibility that guanosine stimulated CO synthesis. Heme oxygenase (HO), the enzyme which synthesizes CO, has been detected in rat adrenal glands [52]. Zinc protoporphyrin-IX (ZnPP) inhibits both the constitutive HO (HO-2) and the inducible HO (HO-1) isoforms of this enzyme [52, 53]. We, therefore, tested the effect of increasing concentrations of ZnPP on the ability of NGF or, NGF plus guanosine to induce neurite outgrowth. Addition of ZnPP (0.01, 0.1, 1 µM) to PC12 cell cultures did not significantly reduce outgrowth of neurites induced by NGF (Figure 3), when compared to control cultures. In contrast, ZnPP attenuated the ability of guanosine to enhance NGF-stimulated neurite outgrowth (Figure 3). As a control for non-specific effects of metalloporphyrins we used copper protoporphyrin-IX (CuPP), which does not inhibit heme oxygenase [54]. The addition of CuPP to PC12 cells did not inhibit either NGF-dependent neurite outgrowth or the ability of guanosine to enhance NGF-dependent neurite outgrowth (data not shown).
Figure 3
Inhibition of heme oxygenase attenuates guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) as described in Figure 1. Some cultures were treated with the selective inhibitor of heme oxygenase zinc protoporphyrin-IX (0.01Y1 µM) for 48 h and the number of cells bearing one or more neurites was determined as described in Figure 1. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Cultures treated with guanosine plus NGF had a significantly (P < 0.01 two-way independent ANOVA) greater proportion of neurite-bearing cells than cultures treated with NGF alone. Zinc protoporphyrin-IX significantly decreased (**P < 0.01) the neurite growth from cultures treated with guanosine plus NGF, but had no significant effect on neurite outgrowth in cultures treated with NGF alone. Data represent the mean ± SEM of 12 determinations from two replicate experiments.
Guanosine induces heme oxygenase-1 (HO-1) expression
PC12 cells have been previously shown to express both HO-1 and HO-2 proteins under basal conditions [55]. Since the addition of ZnPP to PC12 cultures decreased NGF-dependent neurite outgrowth, we attempted to establish whether guanosine treatment had any effect on the expression of the HO-1 protein during the first 48 h of this process. We treated PC12 cultures with guanosine alone (300 µM), or with NGF alone (40 ng/ml), or with a combination of guanosine plus NGF for 48 h, and determined HO-1 expression by Western immunoblot analysis. We used the monoclonal antibody directed against rat HO-1, which recognizes a single protein band, at 32 kDa, the reported molecular weight for this enzyme [36] (Figure 4b). Western immunoblots were quantified and normalized against their corresponding β-actin band. Statistical analysis revealed no differences in β-actin expression for any of the treatments at the different time points (data not shown); therefore, this was used as a control for normalizing HO-1 protein expression. There was no significant change in HO-1 expression in untreated PC12 cells during the 48-h period. In cells exposed to guanosine alone HO-1 expression, unexpectedly, was significantly reduced at 6 h compared to untreated cells at 0 time (P < 0.05), and at 6 h (P < 0.01) (Figure 4). A possible explanation for this result may stem from the observation that HO-1 gene expression is suppressed by elevated intracellular calcium concentrations [56] and guanosine has been shown to increase intracellular calcium concentrations in astrocytes [57]. Since increases in calcium are rapid and transient, this may account for the reduced HO-1 expression at 6 h. The addition of NGF, however, is sufficient to override this effect and so at later time points HO-1 expression is up-regulated by the combination of NGF and guanosine. The addition of guanosine plus NGF significantly increased HO-1 expression at 12 h (P < 0.05), compared to that of control cells at 0 time (Figure 4). After 24 h all three treatments (guanosine alone, or NGF alone or guanosine plus NGF) led to a significant increase in HO-1 expression compared to control cells at 0 time (P < 0.01) (Figure 4). In cells exposed to guanosine alone the enhanced HO-1 expression at 24 h was also significantly different from that detected in untreated cells at 24 h (P < 0.05). Whereas in cells treated with NGF alone, or with the combination of NGF plus guanosine this difference was significant at P < 0.01 (Figure 4). After 48 h, HO-1 expression was elevated significantly only in cells exposed to NGF alone when compared to untreated cells at 0 time, and at 48 h (P < 0.05) for both comparisons. In cells exposed to guanosine alone or to NGF plus guanosine HO-1 expression by 48 h declined and was similar to untreated control values at 0 time and at 48 h.
Figure 4
Guanosine induces the expression of heme oxygenase-1 protein in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C) as described in Figure 2b. The expression of heme oxygenase-1 was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test (○ P < 0.05 compared with control time point 0); (○○ P < 0.01 compared with time point 0); (* P < 0.05 compared with control at same time point); (** P < 0.01 compared with control at same time point). (a) Data represent the mean optical density ± SEM obtained in six independent experiments. (b) Results are representative Western immunoblots obtained in these experiments.
In parallel experiments, we determined whether guanosine had an effect on the expression of the constitutive isoform, HO-2 in these cells. We treated PC12 cultures with guanosine alone (300 µM), or NGF alone (40 ng/ml), or in combination guanosine plus NGF for 48 h and determined HO-2 expression by Western immunoblot analysis. Using the monoclonal antibody directed against rat HO-2 we detected a single protein band, of molecular weight 36 kDa, as described for HO-1 [36] (Figure 5b). Western immunoblots were quantified and normalized against their corresponding β-actin band. Again, statistical analysis revealed no differences in β-actin expression for any of the treatments at the different time points (data not shown), therefore, this was used as a control for normalizing HO-2 protein bands. HO-2 expression was detectable at 0 time in untreated PC12 cells, but none of the treatments had any significant effect on its expression at any time point determined. In the guanosine-treated cells HO-2 expression was elevated slightly at 12 h, but this was not statistically significant.
Figure 5
Guanosine has no effect on heme-oxygenase-2 protein expression in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C) as described in Figure 2b. The expression of heme oxygenase-2 was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANOVA. (a) Data represent the mean optical density T SEM obtained in three independent experiments. (b) Results are representative Western immunoblots obtained in these experiments.
Guanosine increases intracellular cGMP concentrations in PC12 cells during guanosine-enhanced NGF-dependent neurite outgrowth
CO is a known activator of sGC [36]. Since we have shown that HO-1 expression is elevated in PC12 cells exposed to guanosine, we next investigated whether this increased HO-1 expression is accompanied by elevation in intracellular cGMP concentrations. PC12 cell cultures were treated with guanosine alone (300 µM), or NGF alone (40 ng/ml), or in combination guanosine plus NGF for 48 h. Intracellular cGMP concentrations were determined after 6, 12, 24 and 48 h of treatment. In cells exposed to guanosine alone, cGMP concentrations increased significantly after 6 h compared to untreated cells at this time point (P < 0.05). In PC12 cells treated with guanosine plus NGF, cGMP concentrations increased significantly after 6 h compared to untreated cells at 0 time (P < 0.05), remained significantly elevated at 12 h (P < 0.05) and at 24 h (P < 0.05), and declined to values comparable to control by 48 h.
Inhibition of heme oxygenase (HO) attenuates intracellular cGMP concentrations in PC12 cells during guanosine enhanced NGF-dependent neurite outgrowth
Previous experiments have demonstrated that the HO inhibitor, ZnPP, blocked guanosine enhanced NGF-dependent neurite outgrowth of PC12 cells (Figure 3). We have also shown that HO-1 expression and cGMP concentrations were elevated during the first 12–24 h of this neurite outgrowth process (Figures 4 and 6, respectively). We therefore examined whether pre-treatment of cells with the HO inhibitor ZnPP had any effect on cGMP concentrations. Although metalloporphrins, such as ZnPP-IX have been used widely to demonstrate the role for this enzyme in numerous physiological situations, their selectivity for HO has been questioned; as they may also inhibit haemoproteins such sGC and NOS [58]. However, metalloporphrins used at a concentration below 10 µM are more selective for HO [59]. Since we found that ZnPP, at 100 nM concentration significantly attenuated guanosine-enhanced neurite outgrowth (Figure 3), we used this concentration of ZnPP to test its effect on intracellular cGMP concentration. PC12 cell cultures were exposed to guanosine alone (300 µM), or NGF alone (40 ng/ml), or in combination guanosine plus NGF for 12 h. ZnPP (100 nM) was added to some cultures prior to the addition of guanosine, or NGF, or NGF plus guanosine, and intracellular cGMP concentrations were determined. In cells exposed to guanosine alone, or to guanosine plus NGF, cGMP concentrations were significantly elevated compared to untreated cells (P < 0.01) and to cells treated with NGF alone (P < 0.01) (Figure 7). In cultures pretreated with ZnPP, cGMP concentrations were significantly reduced in cells exposed to guanosine alone (P < 0.05), or guanosine plus NGF (P < 0.05) compared to the corresponding treatments without the inhibitor. Treatment of PC12 cells with NGF did not increase intracellular cGMP concentrations, and the addition of ZnPP had no effect on cGMP concentrations.
Figure 6
Guanosine increases cGMP concentrations during guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C) as described in Figure 2b. Cells were lysed at time points 0, 6, 12, 24, and 48 h and cGMP concentrations were determined by a competitive enzyme immunoassay. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test (○ P < 0.05, compared to time point 0); (* P < 0.05, relative to control); (** P < 0.01, relative to control); (Δ P < 0.05, relative to NGF). Data presented represent the mean relative optical density T SEM obtained in six independent experiments.
Figure 7
Inhibition of heme oxygenase attenuates cGMP concentrations during guanosine enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated) fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C) as described in Figure 2b. The selective inhibitor of heme oxygenase, zinc protoporphyrin-IX (100 nM), was added to some cultures prior to the addition of guanosine, or NGF, or guanosine plus NGF. Cells were lysed after 12 h and cGMP concentrations were determined by a competitive enzyme immunoassay. Open bars: no zinc protoporphyrin-IX added, closed bars: zinc protoporphyrin-IX added. Statistical analysis was performed using a one-way ANOVA followed by Fischer's LSD post-hoc comparison test (** P < 0.01, relative to control); (ΔΔ P < 0.01, relative to NGF); (○ P < 0.05, relative to treatment without zinc protoporphyrin-IX). Data presented represent the mean relative optical density T SEM obtained in six independent experiments.
Discussion
Addition of guanosine to cultures of undifferentiated, NGF-naïve PC12 cells modestly increases the proportion of cells with neurites after 48 h and, in cultures treated with maximally effective concentrations of NGF, addition of guanosine produces a significant further increase in the proportion of neurite-bearing cells [15]. This implied that the effects of NGF and guanosine are mediated through distinct signaling systems. We reported previously that, whereas some of the neuritogenic effects of guanosine were mediated by increases in intracellular cAMP [22, 23], there was also a cAMP-independent component [22]. Several points led us to consider that a signaling pathway involving cGMP may be responsible for the cAMP-independent component. Vuorinen et al. [25] had shown that extracellular guanosine increases intracellular cGMP in rat mesenteric artery through a NO-dependent mechanism; nitric oxide donors enhance NGF-dependent PC12 cell neurite outgrowth via a cGMP-dependent mechanism [26]; and the neuritogenic effects of the NO donors are abolished by guanylyl cyclase inhibitors and mimicked by cGMP analogs [26]. Our data indicated that indeed cGMP is involved in the neuritogenic effects of guanosine in PC12 cells, but, surprisingly, that NO is not.
Increases in intracellular cGMP are often due to activation of GC by NO [35, 45]. Schulick et al. [60] demonstrated that activation of GC by NO donors or atrial natriuretic peptide enhanced NGF-dependent neurite outgrowth in PC12 cells. Further, in PC12 cells NGF upregulates NOS expression, and NO plays an important role in their cellular differentiation [32]. However, NGF-induced differentiation of undifferentiated NGF-naïve PC12 cells could not be abolished by inhibitors of sGC, and NO alone is not sufficient to induce PC12 cell differentiation [26, 33].
In the experiments reported here, pre-treating undifferentiated, NGF-naïve PC12 cells with the NOS inhibitor l-NAME, had no effect on the guanosine-enhanced NGF-induced neurite outgrowth. Moreover, like others [61, 62], we found that neither nNOS nor iNOS isoforms were expressed during the first 48 h of cellular differentiation of PC12 cells. Therefore, NO cannot mediate the neuritogenic effects of guanosine on PC 12 cells during the first 48 h.
Rather than NO, our data indicated that CO might activate sGC in response to guanosine in undifferentiated PC12 cells. The heme oxygenase inhibitor, ZnPP attenuated both the ability of guanosine to enhance NGF-dependent neurite outgrowth, and its ability to increase intracellular cGMP, alone or in the presence of NGF. This led us to conclude that guanosine either activated constitutive HO-2 or induced the expression of either HO-1 and/or the HO-2 isoforms of this enzyme.
Our data indicated that, as others have reported [55], both HO-1 and HO-2 were expressed constitutively in PC12 cells under basal cell culture conditions. Guanosine induced a significant increase in the expression of HO-1 protein at 24 h in undifferentiated PC12 cells, but had no significant effect on HO-2 expression. Guanosine, in the presence of NGF, produced an even more rapid and pronounced enhancement of HO-1 expression, which was detectable after 12 h. Treatment of PC12 cells with NGF alone also increased the expression of HO-1. Several authors reported recently that NGF promotes HO-1 gene transcription in PC12 cells [63, 64] via activation of the MEK pathway. HO-1 mediates the anti-oxidative and antiapoptotic effects of NGF in serum-deprived PC12 cells [63]. In PC12 cells exposed to oxidative stress induced by 6-hydroxy dopamine, NGF promotes HO-1 upregulation, in this case via the PI3K/Akt/PKB pathway [64].
Others [63, 64] have reported increased HO-1 protein by 6–9 h after exposure to NGF, whereas in our study we observed no change in HO-1 protein concentration until 24 h after NGF addition. This discrepancy may be due to a difference in the PC12 cell line, or to the experimental conditions to which the cells were exposed.
We did not expect to find inhibition of HO-1 protein expression by guanosine at 6 h. It has been reported however, that the HO-1 gene expression is suppressed by the elevation of intracellular calcium concentration [56]. Since we have found that guanosine increases intracellular calcium concentrations in astrocytes [57], similar mechanisms may downregulate HO-1 expression in PC12 cells at the 6-h time point. As the guanosine-stimulated calcium increases are rapid and transient, at later time points (12–24 h) other intracellular pathways, such as the MAPK and PI-3 kinase pathways are activated, and these are known to promote HO-1 gene expression. Most likely, by 48 h these pathways are no longer activated.
In contrast to HO-1, neither guanosine nor NGF, alone or in combination with guanosine, affected HO-2 expression. This confirms the results of others who have found that HO-2 is not regulated by NGF [63, 64]. The HO-1 isozyme is induced by diverse intracellular signals (reviews: Maines [36], Immenshchuh and Ramadori [65]), that include some of the pathways which activate protein kinases, such as PKA [66], or PKC [67, 68], or the second messenger cGMP [42] most likely via PKG [69]. These pathways likely converge on nuclear effectors such as the nuclear factor-κB (NF-κB), activator protein-1 (AP-1) and heat shock factor, to enhance HO-1 gene transcription by interacting with the regulatory elements in the promoter region the gene [36, 65].
We reported previously that inhibition of transport of guanosine into PC12 cells did not affect its neuritogenic activity [23], raising the possibility that its effects are mediated by activation of a cell surface, membrane-bound receptor. Indeed, recently we have identified and pharmacologically characterized a specific binding site for guanosine in rat brain membranes [70], in cultured astrocytes [71] and in PC12 cells (F. Caciagli et al., unpublished observations), with characteristics of a Gi-protein coupled receptor [5, 70]. Occupation of this putative receptor by guanosine activates the MAPK pathway [5] and the PI3K/Akt/PKB pathway [72, 73]. These effects are most likely mediated by the β,γ-subunit of the Gi-protein, coupled to the guanosine receptor [74–76]. Guanosine has also been shown to increase intracellular cAMP concentrations in several cell types [22–24] and phosphorylate the cAMP binding protein, CREB (F. Caciagli et al., unpublished observations).
In PC12 cells NGF-promoted induction of HO-1 expression requires the activation of the MEK/ERK pathway [63] and the PI3K/Akt/PKB pathway [64]. Since guanosine, via its putative receptor may also stimulate these intracellular pathways, HO-1 expression in PC12 cells may be regulated by similar mechanisms.
Guanosine plus NGF in combination induce HO-1 expression to a similar extent than NGF alone at 6, 12 and 24 h. At 48 h, however the effect of guanosine plus NGF on HO-1 expression is comparable to that of control and lower than NGF alone. Since the mechanisms that induce HO-1 expression are complex, the roles played by guanosine and NGF, and the time course of these effects are unclear. We have shown, however that guanosine alone is insufficient to cause major neurite outgrowth. It does however enhance the NGF-induced neuritogenesis. Neuritogenesis is not solely due to HO-1 expression. Either HO-1 or HO-2 could be responsible for these early events since ZnPP, a non-selective HO inhibitor reduces the concentration of cGMP elicited by guanosine or guanosine plus NGF by 12 h.
NGF activates a number of signalling pathways in PC12 cells, including the MAP kinase and PI3-kinase pathway [18] and the c-JUN N-terminal kinases (JNKs.) [20]. Induction of the HO-1 gene by these pathways seems to override the inhibitory effect of guanosine on HO-1 expression. HO-1 expression at 12 and 24 h is upregulated to a similar extent by guanosine alone, or by NGF alone or by guanosine plus NGF, so there is no additive effect of the combination treatment. As the mechanisms involved in HO-1 gene induction are complex and not clearly defined, our results suggest that the activation of these processes by the three treatments is comparable.
At 48 h HO-1 expression in cells treated with guanosine alone, or guanosine plus NGF is similar to control. In the NGF-treated cells, however HO-1 expression is still significantly upregulated compared to control. We attribute this to a difference in the time course of HO-1 gene activation by guanosine, and NGF. Since NGF-activated intracellular signalling pathways are complex and manifold [18, 20] these effects may persist for a longer time. These results are different from those we obtained at 6 h. At this time point, the only significant effect on HO-1 expression is its inhibition by guanosine alone, and this may be due to downregulation of HO-1 expression by guanoisne-stimulated elevations in intracellular Ca2+ concentrations as described above.
In several types of cells, HO-1 expression is regulated by the NO-cGMP pathway (reviewed by Maines [36]). In the case of guanosine this mechanism is not likely to play a significant role, as neither iNOS, nor nNOS was detected in NGF-treated PC12 cells during the first 48 h of guanosine-promoted neurite outgrowth.
In contrast, upregulation of HO-1 expression by activation of the constitutive HO-2 via the transcription factor NF-κB, is a very plausible alternative mechanism [36]. HO-2 may be activated by PKC, which in turn is stimulated by increased intracellular calcium concentrations [77]. PKC may also activate sGC directly, by phosphorylation, thus providing a receptor-mediated elevation of cGMP concentrations in PC12 cells [78]. We have shown that guanosine increases intracellular calcium concentrations under certain conditions [57], and this in turn may activate PKC. Thus, guanosine may activate HO-2 in a calcium-dependent manner. The time course of intracellular cGMP elevation elicited by guanosine, or by guanosine plus NGF, is consistent with this hypothesis. In PC12 cells exposed to guanosine alone, or to guanosine and NGF, cGMP concentrations were significantly elevated by 6 h and until 12 h in cells exposed to guanosine plus NGF. Although the addition of NGF to PC12 cells induces HO-1 expression at 24 and 48 h, this treatment does not increase cGMP concentrations. Initially we were also puzzled by these data, as we expected a parallel increase in the cGMP concentrations with this treatment. It has been reported by Liu et al. [79] that NGF treatment of PC12 cells rapidly decreases sGC mRNA and protein in a transcription- and translation-dependent manner. So our interpretation of these results is that although HO-1 protein is upregulated by NGF, there is a concurrent suppression of sGC expression, so no increase in cGMP is detected under these conditions. In addition, NGF may also inhibit the activities of some phosphodiesterase isoforms, and thus decrease intracellular cGMP concentrations [80].
Since HO-1 expression in the guanosine treated cells was not observed until 24 h, and since neither iNOS nor nNOS was detectable at these times, our data is most parsimoniously explained by activation of HO-2.
Although we have not determined HO-2 activity, we have shown, using Western immunoblot analysis that this isozyme is expressed in PC12 cells constitutively, throughout the time course of the experiment (from 0 to 48 h) during all treatment conditions.
CO synthesized by HO-1 and HO-2 will bind and activate sGC, increasing intracellular cGMP [35–38] and this in turn may activate protein kinase G (PKG) [81, 82] and regulate phosphodiesterases (PDE) [81, 82]. Our data are compatible with the neuritogenic effects of guanosine being mediated by cGMP, possibly from early activation of HO-2, and later the induction of HO-1, leading to CO generation, and activation of sGC. But how these molecular events enhance neuritogenesis is unknown. CO itself may directly activate several intracellular signaling pathways, including the MAPK pathway [83] and p38 MAPK [84], the transcription factor NF-κB [85] and induce the transcription of immediate early genes [37, 38].
Recently, Xiang et al. [86] proposed that navigation of growing axons in response to guidance cues, is mediated by Gi protein coupled receptors, via elevation of cytosolic cGMP [86]. This is another mechanism by which guanosine may promote differentiation of PC12 cells.
In conclusion, we have demonstrated the importance of HO, CO and cGMP in mediating the neuritogenic effect of guanosine. The relationship of this signaling mechanism to the putative Gi-protein-linked receptor for guanosine is currently the subject of active investigation in our laboratories.

guanosine
neurite outgrowth
pc12 cells
heme oxygenase
cyclic gmp
ngf
carbon monoxide
nitric oxide synthase