Introduction

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Guanylyl cyclase enzymes are found throughout the body and catalyze the synthesis of the second messenger cGMP from GTP. They exist in two broad families, membrane-bound (particulate) and soluble. The particulate cyclases serve as peptide (or putative peptide) receptors, whereas soluble guanylyl cyclase (sGC) functions as the major receptor for nitric oxide (NO).

cGMP elicits its many acute physiological effects by influencing the activity of kinases, phosphodiesterases, and ion channels (Lucas et al., 2000). In the longer term, the molecule may also be of relevance to pathophysiological situations, as either a protectant or a mediator of damage. For example, a neuroprotective effect of cGMP has been shown in cerebellar slices (Garthwaite and Garthwaite, 1988), spinal cord (Weill and Greene, 1984; Urushitani et al., 2000), and developing retinal explants (Guimaraes et al., 2001). Conversely, cGMP has been implicated in pathological cascades in the retina (Tsang et al., 1996), cortical neurons (Frandsen et al., 1992), and elsewhere.

Degenerative disorders in the central nervous system affect both gray and white matter. White matter, containing myelinated axons and glial cells, is selectively affected in several conditions, including stroke, trauma, and multiple sclerosis (Stys, 1998; Trapp et al., 1998). Much less is understood about the mechanisms of damage to white matter relative to neuronal cell bodies residing in the gray matter. A convenient model for white matter pathology is the isolated optic nerve preparation, and studies of the effects of metabolic stress (anoxia and simulated ischemia) have consistently suggested that Na⁺ entry through voltage-dependent ion channels leading to lethal Ca²⁺ influx is the primary mechanism (Stys, 1998). Evidence from investigations of spinal cord trauma in vivo lends support to this proposal (Rosenberg et al., 1999).

Another putative mediator of damage to numerous tissues, including CNS white and gray matter, is NO, which is produced by constitutive or inducible NO synthases. In the case of white matter, circumstantial evidence indicates that excessive NO production contributes to damage occurring in multiple sclerosis, glaucoma, acquired immunodeficiency syndrome dementia, and diabetic neuropathy (Bo et al., 1994; Stevens, 1995; Neufeld et al., 1997; Rostasy et al., 1999). However, it remains unclear whether the NO is a cause or an effect of these diseases and whether NO synthase up-regulation or induction in these pathological conditions has a deleterious or ameliorating effect (Willenborg et al., 1999). Hitherto, there have been relatively few studies exploring the potential toxicity of NO toward cells and axons in white matter (Merrill et al., 1993; Redford et al., 1997). In support of this possibility, we have recently found that, when applied to the isolated optic nerve, NO is capable of killing both axons and glial cells, with the former being more vulnerable (Garthwaite et al., 2001). Interestingly, optic nerve axons are also rich in sGC and exposure to NO leads to the apparently exclusive accumulation of cGMP in these elements (Garthwaite et al., 1999b). This raises the question as to whether cGMP plays any role (protective or destructive) in the axon pathology. The initial aim of the present experiments was to examine this possibility by using pharmacological and other tools to manipulate cGMP levels. In the course of these studies, we made the observation that an allosteric activator of sGC, 3-(5-hydroxymethyl-2-furyl)-1-benzyl-indazole [known as YC-1; Fig. 1; Ko et al., 1994; Wu et al., 1995], exerted a powerful protective effect on NO toxicity toward axons and the subsequent experiments were geared toward identifying the underlying mechanism.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structures of YC-1 (A) and sipatrigine (B).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Optic Nerves. The method has been described previously (Garthwaite et al., 1999a). Briefly, Wistar rats (220-260 g) were decapitated as approved by the British Home Office and the local ethics committee. The nerves were quickly excised and incubated in 50-ml flasks containing 20 ml of an artificial CSF (aCSF) solution composed of 120 mM NaCl, 2.0 mM KCl, 2.0 mM CaCl₂, 26 mM NaHCO₃, 1.18 mM KH₂PO₄, 1.19 mM MgSO₄, and 11 mM glucose, continuously gassed with 95% O₂/5% CO₂. The flasks were held in a shaking water bath at 37°C and left to incubate for 1 to 1.5 h before the experiments were started.

Histology. The nerves were fixed, embedded in resin, sectioned (1 µm), and stained with toluidine blue by using conventional techniques. Morphometric analysis of the axonal damage was performed using an image analysis system as described previously (Garthwaite et al., 1999a). Briefly, for each nerve, the mean internal Feret diameter of axons was measured in four fields, each having an area of 2500 µm². The numbers of axons with values above 2.5 µm/10⁴ µm² provided the "axonopathy index". With this cutoff, the majority of undamaged thicker axons are excluded such that, in control nerves, only about 1.5% of all axons are registered in the index. Mean data were derived from four to eight nerves in two to four separate experiments. For each test condition in a given experiment, there were two to three nerves, each from a different animal.

cGMP Measurement. Sister nerves of those used to examine PAPA/NO toxicity were exposed to PAPA/NO for 30 min, a period that does not cause damage (Garthwaite et al., 2001), in the absence or presence of the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), the inhibitor of sGC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or YC-1, as detailed in the text. In some experiments aimed at determining concentration-response curves for PAPA/NO and YC-1 on sGC (Fig. 3B), the exposure period to PAPA/NO was reduced to 5 min to limit possible desensitization of sGC and other associated proteins. At the end of the exposures, the nerves were inactivated by boiling in hypotonic buffer, homogenized by sonication, and the cGMP and protein levels measured by radioimmunoassay and the bicinchoninic method, respectively (Garthwaite et al., 1999b).

Preparation of Synaptosomes. This was done essentially as described by Pauwels et al. (1986). Cerebral cortices dissected from Male Wistar rats (200-300 g) were homogenized in 9 volumes of ice-cold 0.25 M sucrose by using a motor-driven, glass-Teflon homogenizer (Potter S; B. Braun, Allentown, PA) using eight up and down strokes at 900 rpm. The homogenate was centrifuged at 1036g at 4°C for 10 min and the supernatant collected. The remaining pellet was resuspended as described above in fresh ice-cold 0.32 M sucrose and the centrifugation step repeated. The supernatant fractions were pooled and centrifuged at 46,000g for 15 min. The resulting pellet was resuspended in assay buffer at a final concentration of 10 to 20 mg of cortex/ml, wet weight.

Veratrine-Evoked Uptake of [¹⁴C]Guanidine. Test compounds, veratrine (100 µg/ml final concentration), and synaptosomes (4 mg/ml, wet weight) were incubated in the absence or presence of tetrodotoxin (TTX; 1 µM) at 37°C for 5 min in polypropylene test tubes. Uptake was initiated by the addition of prewarmed [¹⁴C]guanidine (final concentration 1 µCi/ml) and stopped 2 min later by the addition of 10 ml of ice-cold wash medium as described by Pauwels et al. (1986). Incubates were immediately filtered under vacuum through GF/C filters by using a Brandel harvester. The incubation tubes were rinsed with 5 ml of ice-cold wash buffer, which was then used to wash the filter. Filters were transferred to minivials (Beckman Coulter, Fullerton, CA) with the use of a Brandel deposit/dispense system and subsequently counted by liquid scintillation spectroscopy with Picofluor⁴⁰ liquid scintillator.

Batrachotoxinin-B (BTX-B) Binding. This was carried out using the method described by Catterall et al. (1981), except that both bovine serum albumin and TTX were omitted from the incubation medium. Binding was initiated by the addition of synaptosomes (final concentration 10 mg/ml, wet weight) to a mixture of test compound and 10 nM [³H]BTX-B in the absence or presence of scorpion venom (25 µg/ml final concentration). Samples were mixed and incubated for 90 min at 25°C. Ice-cold wash medium (5 ml) was added and then the samples subjected to vacuum filtration through GF/C filters by using a Brandel harvester. Incubation tubes were rinsed with 5 ml of ice-cold wash buffer, which was then used to wash the filter. Radioactivity in the filter was counted as described above.

Whole-Cell Recording from Dissociated Hippocampal Neurons. Wistar rats (14-21 days old) were decapitated and the brain was quickly removed and placed into an ice-cold solution containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl₂, 1.0 mM NaH₂PO₄, 26.2 mM NaHCO₃, and 11 mM glucose, bubbled with 95% O₂/5% CO₂. The hippocampi were dissected and 400-µm slices were cut using a vibrotome. Slices were maintained in a holding chamber containing the same solution at room temperature for at least 1 h before use. For preparation of the dissociated neurons, two to three slices were placed in oxygenated PIPES buffer solution that contained 86 mM PIPES, 30 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 10 mM glucose at 37°C with added protease XXIII (3 mg/ml). After 10 min, slices were transferred to PIPES buffer at room temperature containing trypsin inhibitor and bovine serum albumin (both at 1 mg/ml). A portion of the CA1 region was removed using a micropunch and cells dissociated by gentle trituration with fire-polished Pasteur pipettes. The cell suspension was then left on a poly(L-lysine)-coated coverslip for about 15 min.

Optic Nerve Compound Action Potentials. The grease-gap recording technique described previously was used (Garthwaite et al., 1999a). Briefly, the nerve was placed in a three-compartment recording chamber. The central portion of the nerve was positioned in the middle chamber with the ends passing through greased holes in the partitions. In the first compartment, a bipolar stimulating electrode was positioned on the surface of the nerve. Compound action potentials were elicited at a rate of 0.2 Hz by using square-wave voltage pulses of 60- to 90-µs duration and 10- to 15-V amplitude. To reduce the appearance of biphasic action potentials on the recording, the length of the nerve entering the third compartment was kept as short as possible. All three chambers were filled with aCSF and were heated to 37°C by a water jacket. The central compartment was perfused with aCSF at a rate of ~1.5 ml/min. The outer compartments were not perfused. The YC-1 was applied in the perfusate diluted 3:1000 from a 10 mM stock solution. The compound action potential was monitored differentially using Ag/AgCl electrodes embedded in agar, amplified (Grass P16; Grass Instruments, Quincy, MA), and recorded on a personal computer by using Clampex 8 (Axon Instruments) with a sampling rate of 200 kHz. The traces shown are averages of five sequential responses obtained before drug application and before washout. The area under these averaged waveforms was calculated using Clampfit 8 (Axon Instruments).

Statistical Analysis. Results are given as means ± S.E.M. and were evaluated using Student's t test for unpaired variables (two-tailed), with P < 0.05 being considered significant.

Materials. PAPA/NO was from Alexis Corporation (Bingham, Nottingham, UK). TTX was from Latoxan Laboratories (Rosans, France). Sipatrigine was supplied by the Wellcome Research Laboratories (Beckenham, Kent, UK). [¹⁴C]Guanidine hydrochloride (2.18 GBq/mmol) was obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK), and batrachotoxinin-A 20--benzoate, [benzoyl-2,5-³H] (1258 GBq/mmol) from PerkinElmer Life Sciences (Boston, MA). YC-1 was provided by the chemistry department of the Wolfson Institute for Biomedical Research. Other chemicals were from Sigma-Aldrich (Poole, Dorset, UK), BDH/Merck (Poole, Dorset, UK), or Tocris-Cookson (Bristol, UK).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

cGMP in NO-Induced Axonopathy. In agreement with previous findings (Waxman et al., 1992; Garthwaite et al., 1999a), histology of optic nerves incubated under control conditions showed that they contained well preserved axons and glial cells (Fig. 2A). Exposure of the nerves to 1 mM PAPA/NO for 2 h (plus 2-h recovery) resulted in selective axonal degeneration characterized by persistent swelling (Fig. 2B). It has been shown previously that this damage is irreversible, inhibited by the NO scavenger oxyhemoglobin, and is not observed using PAPA/NO depleted of its NO moiety (Garthwaite et al., 2001). Quantitation of the damage was carried out using a morphometric method that gave an "axonopathy index" corresponding to the numbers of axons having an internal Feret diameter of greater than 2.5 µm/unit area (10⁴ µm²). PAPA/NO treatment caused an approximately 10-fold increase in this index (Table 1).

View larger version (169K): [in this window] [in a new window]   Fig. 2.   Light micrographs illustrating axonoprotective effect of YC-1 in rat isolated optic nerve subjected to NO toxicity. A, cross section of a control nerve maintained in vitro for 6 h. In A to E, arrowheads indicate axons and arrows indicate glial cells. B, nerve incubated with 1 mM PAPA/NO for 2 h followed by 2-h recovery in normal incubating media, showing swollen axons. C, nerve incubated with 1 mM PAPA/NO for 2 h in the presence of 30 µM YC-1, showing no damage. D, nerve subjected to 1 h OGD followed by 2-h recovery showing swollen axons. E, nerve subjected to 1 h of OGD in the presence of YC-1 (30 µM), showing significant preservation of axonal morphology. Scale bar (in C, but applies to A-E), 10 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Relative potencies of YC-1 for inhibiting NO-induced axonopathy (A) and potentiating NO-stimulated sGC activity (B). In A, the nerves were incubated with PAPA/NO for 2 h followed by 2-h recovery, in the presence or absence of different concentrations of YC-1. *P < 0.01, **P < 0.00001 versus PAPA/NO control; ns, not significant versus control nerves (); n = 4 to 8. In B, the effect of YC-1 on cGMP levels was measured in nerves (n = 4) exposed for 5 min to PAPA/NO (1 µM) in the presence of IBMX (1 mM added 15 min beforehand). YC-1 was added 10 min before PAPA/NO. The inset shows the concentration-cGMP response curve for PAPA/NO in the presence of IBMX (n = 3). All data represent means ± S.E.M.

YC-1 against OGD-Induced Axonopathy. To investigate whether the protective effect of YC-1 extended to other insults, its effects against OGD were tested. Exposure of the nerves to 1 h of OGD followed by 2-h recovery caused severe swelling and distension of the axons, an effect that was qualitatively and quantitatively similar to that produced by PAPA/NO (Figs. 2D and 4). When the nerves were incubated with YC-1 (30 µM) during 1-h OGD, axons were largely spared (Figs. 2E and 4). In the same experiments, the Na⁺ channel blocker sipatrigine (100 µM; Fig. 1; formerly known as BW619C89), which had previously been shown to protect against OGD and NO toxicity in the optic nerve (Garthwaite et al., 1999a, 2001), preserved the axons to a degree not significantly greater than that afforded by YC-1 (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Protection of axons by YC-1 against PAPA/NO toxicity and OGD in rat optic nerve resembles that of Na+ channel inhibitors. Nerves were exposed to PAPA/NO (1 mM; 2 h) or OGD (1 h) and then allowed to recover in normal incubation medium for 2 h. TTX (1 µM), sipatrigine (100 µM), YC-1 (30 µM), or Na+-free medium were applied 15 min beforehand and remained for the first 15 min of recovery. Data are means ± S.E.M; n = 4 to 8. *P < 0.00001 versus PAPA/NO control; , P < 0.0001, , P < 0.00001 versus OGD control.

Is Axonoprotective Effect of YC-1 Caused by Na⁺ Channel Inhibition? In agreement with other recent findings (Garthwaite et al., 2001) PAPA/NO toxicity toward axons in the present experiments was inhibited by the Na⁺ channel blockers TTX (1 µM) and sipatrigine (100 µM), and by removing extracellular Na⁺ (Fig. 4), confirming the important role played by the entry of Na⁺ through voltage-sensitive channels in NO-induced axonopathy. With all these interventions, the protection was complete and that given by YC-1 (30 µM) was not significantly different. The possibility that YC-1 is a Na⁺ channel blocker was therefore examined.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves for YC-1 and sipatrigine in assays for Na+ channel inhibition. A, veratrine-evoked uptake of [14C]guanidine into rat cerebral cortex synaptosomes. Compounds under test were preincubated with synaptosomes for 5 min in the presence of either veratrine alone or veratrine plus 1 µM TTX before the addition of [14C]guanidine. B, binding of [3H]BTX-B to rat cerebral cortex synaptosomes. Compounds under test were incubated with rat cerebral cortex synaptosomes and either [3H]BTX-B or [3H]BTX-B plus scorpion venom (SV) for 90 min. In both cases, data are from one representative experiment (of three) and are the means of duplicate measurements at each concentration. IC50 values were computed for the major components of binding and uptake by using the single-site logistic equation, to which the data were fitted (curves).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Electrophysiological analysis of the effect of YC-1 on voltage-dependent Na+ channels. A, voltage-dependent inhibition of Na+ currents in hippocampal neurons. Currents were elicited using a switched prepulse protocol: one test pulse depolarized from a membrane potential (Vh) of 90 mV to 0 mV for 10 ms; the second was preceded by a prepulse to 60 mV for 30 s, as shown beneath each current trace. Sample traces are from the same cell under control conditions and in the presence of YC-1 (30 µM). Bar graph shows mean data from four cells; ns, not significant; *, P < 0.01 versus control. B, YC-1 (30 µM) did not affect compound action potentials in optic nerve. Sample traces are averages of five consecutive sweeps. Bar graph shows mean data from four nerves; ns, not significantly different from control.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The identification of molecules able to protect CNS tissue at risk in acute and chronic neurodegenerative disorders, with known mechanisms of action, is a goal of potentially great significance for understanding the pathogenesis of those disorders and for the development of new treatments. In the present study, we have serendipitously found a novel chemical species that is able to protect CNS white matter axons to a remarkable degree, and have attempted to discover how it produces this effect.

Role of cGMP. In light of evidence that cGMP can be protective or detrimental to cell survival, together with the finding that NO evoked a large and selective accumulation of cGMP in the principal targets of its toxicity in optic nerve (the axons), the first aim was to investigate whether this response had any relevance to the pathology. Manipulation of cGMP accumulation over a wide range by using inhibitors of sGC and phosphodiesterase enzymes, or addition of an exogenous cGMP derivative, however, had no influence on the PAPA/NO-induced axonopathy. This indicates that the two effects are not causally related, at least so far as the relatively acute pathology studied here is concerned.

Protection of Axons by YC-1. The abolition of NO toxicity by YC-1 could not be explained by the attendant increase in cGMP accumulation because inhibition of this response by ODQ or augmenting it with IBMX had no effect. Consequently, an alternative mechanism of action was sought. Several similarities exist between NO toxicity and the injury induced by anoxia or OGD with respect to optic nerve axons, notably the joint dependence on extracellular Ca²⁺ and Na⁺ concentration and blockade by Na⁺ channel inhibitors such as TTX (Stys, 1998; Garthwaite et al., 1999a, 2001). This led to the suggestion that NO kills axons by inhibiting mitochondrial respiration, leading to metabolic inhibition (Garthwaite et al., 2001). The common underlying mechanism is likely to be the one first proposed for anoxia-induced axonopathy (Stys et al., 1992). According to this hypothesis, metabolic stress results in excessive influx of Na⁺ through voltage-sensitive Na⁺ channels. Of particular importance here may be the slowly inactivating Na⁺ channels that are prominent in optic nerve axons. Loading of the axoplasm with Na⁺ results in a reversal of the Na⁺-Ca²⁺ exchanger, leading to a secondary influx of Ca²⁺, which causes the irreversible axon damage. That Na⁺ channel inhibitors provide a high degree of protection toward axons in these conditions suggested a plausible mechanism of action of YC-1, a possibility supported by the finding that the compound also protected against OGD-induced axonal damage in the optic nerve. In accordance with this possibility, in several assays, the compound was found to be a Na⁺ channel inhibitor.

Implications. In terms of its chemical structure (Fig. 1), YC-1 does not seem to resemble sipatrigine or indeed any other known inhibitor of voltage-dependent Na⁺ channels. Although other potential applications of such a molecule (e.g., as an anticonvulsant, analgesic, and protectant for gray matter) need to be explored, compounds able to protect white matter from damage without interfering with normal nerve function are rare. Indeed, only sipatrigine, which has undergone early clinical trials for stroke (Muir et al., 2000), has so far shown such a profile of activity. The other activities of YC-1 on sGC and phosphodiesterases might be undesirable in the setting of neuroprotection in stroke, because they would cause a reduction in blood pressure (Rothermund et al., 2000). Nevertheless, YC-1 could serve as a prototype for the synthesis of more selective Na⁺ channel inhibitors that would have potential as therapy for degenerative disorders affecting white matter axons, such as glaucoma and spinal cord injury, and possibly those affecting gray matter as well. Finally, the identification of this additional mechanism of action of YC-1 has implications for the growing use of this agent to probe sGC-related functions, as it now becomes necessary to ensure that any effects seen are not caused, or complicated, by Na⁺ channel inhibition. With the development of structural analogs of YC-1 for possible clinical use (Stasch et al., 2001), this issue assumes increasing importance.