1 Division of Neurology, Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan
2 Division of Nephrology and Diabetes, Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan
3 Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan
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ABSTRACT |
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Diabetic neuropathy is the most common disease of peripheral neuropathy in Western countries, as well as the most frequent microangiopathic complication of diabetes (1). Some patients with diabetic neuropathy have various forms of neuropathic symptoms, including hyperalgesia and spontaneous pain, which are often developed in early stages but may occur at any stage. The painful symptoms are troublesome and reduce the patients quality of life. Thus, the relief from painful symptoms should be a main purpose for the treatment of diabetic neuropathy. However, the mechanism by which such neuropathic symptoms develop remains unclear, and useful drugs for pain management in diabetes remain unavailable.
The underlying mechanisms of painful symptoms may be closely associated with hyperglycemia and/or the pathogenic mechanism of diabetic neuropathy itself. Various hypotheses have been proposed to explain the pathogenesis of diabetic neuropathy (2,3): polyol pathway hyperactivity, decreased nerve blood flow followed by endoneurial hypoxia, increased glycation of proteins, abnormal activity of protein kinase C (PKC), decreased neurotrophism, and the associated exaggeration of oxidative stress. Among these hypotheses, the involvement of PKC may be one of the most relevant. Hyperglycemia activates PKC, especially its ßII isoform, through increased de novo synthesis of diacylglycerol in retina (4), glomeruli (5), and aorta and heart (6). This increased activity of PKCß may impair retinal (4) and endoneurial (7) blood flow, causing renal hyperfiltration (8), resulting in the development of diabetic retinopathy, nephropathy, and neuropathy. In addition, selective PKCß inhibitors ameliorate these abnormalities (7,9,10).
The role of PKC hyperactivity has also been well investigated with reference to pain generation, using not only phorbol esters and PKC activators (11), but also various members of the PKC superfamily. Chronic inflammation-evoked thermal hyperalgesia may involve several protein kinases, including PKC and protein kinase A (12). PKC
has also been shown to regulate nociceptor function in the experiments using either PKC
mutant mice or a PKC
-selective inhibitor peptide in dorsal root ganglion (DRG) neurons (13). Increase in PKCßII activity has been reported to participate in hyperalgesia caused by adjuvant-induced inflammation in the rat hind paw (14).
The important contribution of PKC to hyperalgesia has also been reported in diabetic animals. Phorbol esters enhance thermal hyperalgesia in diabetic mice. The hyperalgesia and C-fiber hyperexcitability to mechanical stimuli observed in diabetic rats are reduced by intradermal injection of agents that inhibit PKC (15). In an in vitro study using rat sensory neurons, PKC was shown to mediate release of substance P and calcitonin gene-related peptide (CGRP) from sensory neurons. This PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia (16). Thus, although the hyperactivity of PKC is thought to contribute to hyperalgesia in diabetes, the responsible mechanism has not yet been identified.
Aside from the implication of PKC in the generation of pain in diabetes, attenuated neuronal nitric oxide synthase (nNOS)-cGMP system in DRGs may play a role in the pathogenesis of hyperalgesia in streptozotocin (STZ)-induced diabetic rats (17). In addition, tetrodotoxin (TTX)-resistant (TTX-R) Na+ currents are exaggerated in the small DRG neurons of diabetic rats (18); TTX-R Na channels have been considered to profoundly contribute to nociception (19). In spite of the accumulating data on diabetic hyperalgesia and its mechanisms, all of the data so far published have been somewhat fragmentary, so the whole story on diabetic hyperalgesia needs more clarification.
In the present study, we have attempted to examine whether PKCß inhibition may ameliorate hyperalgesia in diabetes and, if so, whether the effect is obtained through nonvascular action on DRG neurons or nerve fibers with special reference of the nNOS-cGMP system and TTX-R Na+channels.
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RESEARCH DESIGN AND METHODS |
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Nociceptive tests.
The mechanical threshold for the nociceptive flexion reflex elicited by stimulation of the dorsal surface of the hind paw was quantified using an analgesymeter (Ugo Basile). This device generates a mechanical force that increases linearly with time. The force was applied by a dome-shaped plunger (1.4 mm diameter, radius of curvature 36°). The nociceptive threshold is defined as the force, in grams, at which the rat withdraws its paw. The average threshold of three training trials constituted the baseline nociceptive threshold for that day. Rats were trained in the paw-withdrawal test at 5-min intervals for 30 min each day for 3 days. In the first experiment, withdrawal threshold was tested in untreated and LY333531-treated diabetic and control rats (n = 9 per group) at 0, 2, 4, and 6 weeks after STZ injection. In the second experiment to analyze the effect of L-arginine, nociceptive threshold was compared in rats (n = 5) treated only with saline at 0, 2, 4, and 6 weeks after STZ injection. To evaluate the possible effect of topical PKC inhibition in diabetic hyperalgesia, nociceptive threshold was assessed 6 weeks after STZ injection in the hindpaw of diabetic and age-matched control rats at intervals up to 24 h after intradermal injection of various concentrations of LY333531 into the footpads. The effect was compared with at the contralateral side, where the same volume of saline was injected. LY333531 dissolved in 0.01% DMSO in saline was injected in a volume of 2.5 µl with a 30-gauge needle.
Immunoblotting.
Animals of each group were killed by decapitation under anesthesia. Bilateral L4-6 DRG samples from individual rats were dissected out and immediately homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mmol/l Tris-HCl, pH 7.4; 150 mmol/l NaCl; 1 µg/ml each of leupeptin, aprotinin, and pepstatin; 1 mmol/l EDTA; 1 mmol/l phenylmethylsulfonyl fluoride; 1 mmol/l Na3VO4; 1 mmol/l NaF; and 1% NP-40). Samples were centrifuged at 15,000g for 30 min at 4°C. Proteins in the supernatant were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was incubated with affinity-purified rabbit polyclonal antibodies: PKC (sc-208), PKCßI (sc-209), PKCßII (sc-210) (Santa Cruz Biotechnology), nNOS (Transduction Labs), and monoclonal antibodies to TTX-R Na channel protein, SNS/PN3, and SNS2/NaN (20) (courtesy of Roche Bioscience) after blocking with 5% milk at 4°C overnight.
After standard washing, immunoreactivity was detected by enhanced chemiluminescence on film. The immunoreactive bands were measured using NIH Image.
Membrane preparation and assay for PKCß activity.
Both control and STZ rats were killed by decapitation under anesthesia. Bilateral L4-6 DRG samples from individual rats were dissected out and immediately homogenized in ice-cold buffer A (20 mmol/l Tris-HCl, pH 7.4; 0.25 mol/l sucrose; 0.15 mol/l NaCl; 25 µg/ml each of leupeptin and aprotinin A; 5 mmol/l EDTA; 2.5 mmol/l EGTA; and 2 mmol/l dithiothreitol) using a Wheaton-33 homogenizer for 30 s. Samples were centrifuged for 1 h at 100,000g at 4°C. The supernatant constituted the cytosolic PKC preparation. The pellet was rinsed twice in the same buffer, resuspended by a brief 30-s rehomogenization in buffer A containing 0.5% Nonidet P-40, and incubated on ice for 30 min with intermittent mixing. The extract was then centrifuged at 100,000g for 1 h at 4°C with the supernatant constituting the membrane-associated PKC preparation. The activity of PKCß in both membrane and cytosol fractions was quantified by immunoprecipitation of the isozyme in the resuspended immunocomplexes. PKCßII isoforms in solubilized membrane and cytosol suspensions (100 µg protein/assay) were immunoprecipitated with protein A agarose and affinity-purified rabbit polyclonal IgG antibodies for PKCßII (Santa Cruz) at 3 µg/ml. After overnight incubation at 4°C, immunocomplexes were recovered by brief low-speed centrifugation. They were then rinsed once with a 1:1 mixture of the tissue homogenization buffer and 0.4 mol/l NaCl and recentrifuged for 20 s. The resulting pellet was resuspended in homogenization buffer (25 µl), and PKC activity was quantified using a commercial PKC enzyme detection system (RPN.77; Amersham Life Sciences, Arlington Heights, IL), based on the modification of a mixed micelle assay using a phorbol ester (21).
Immunocomplex assay for phospho-PKC.
To examine the effect of LY333531 on PKC activity in DRGs of diabetic rats, the activities of PKCßII in DRGs were quantified by immunoprecipitation of the isozyme followed by phosphorylated PKCß content in the resuspended immunocomplexes. PKCßII in DRG, homogenized as described above, were immunoprecipitated with protein A agarose and affinity-purified rabbit polyclonal IgG antibody for PKCßII (Santa Cruz) at 1 µg/ml. After 2-h incubation at 4°C, immunocomplexes were recovered by brief low-speed centrifugation and rinsed once with homogenized RIPA buffer. The resulting pellet was resuspended in sample buffer and expanded to SDS-PAGE. After transferring to nitrocellulose membrane, the membranes were incubated with rabbit polyclonal phospho-PKC antibody (no. 9371S; New England Biolabs). Immunoreactivity of phospho-PKCß was detected as described above. The PKCßII protein was confirmed by reblotting the molecule of the band corresponding to phospho-PKCßII.
cGMP and cAMP assay.
Bilateral L4-6 DRG samples were dissected out, immediately homogenized in 0.1N HCl, and heated at 100°C for 10 min in Eppendorf test tubes. The supernatant was obtained after centrifugation at 15,000g for 30 min. The cGMP content of the solution was measured using a cGMP radioimmunoassay kit (Yamasa). Protein in the solution was assayed, after neutralization, by the Bradford method (Bio-Rad). The cGMP content was corrected for protein concentration.
Effect of LY333531 on cGMP content of DRG: ex vivo assay.
Bilateral L4-6 DRGs were dissected out from control and diabetic rats and immediately incubated in the oxygenized PBS medium (pH 7.4) containing 500 mmol/l isobutyl methyl xanthine and phosphodiesterase inhibitor for 30 min at 37°C. Our preliminary data suggested that the maximum effect of LY333531 on DRG cGMP content was obtained at 200 µmol/l LY333531 (data not shown). One side of L4-6 DRG for each rat was incubated in the medium without LY333531 and the other side was incubated in the presence of 200 µmol/l LY333531. After incubation, cGMP content of DRGs was measured as described above.
Immunohistochemistry.
Animals were deeply anesthetized (pentobarbital, 60 mg/kg, intraperitoneally) and perfused intracardially with PBS followed by paraformaldehyde fixative. After perfusion, L4-6 DRGs were removed and fixed by immersion for 1216 h at 4°C. Tissues were then rinsed in PBS, cryoprotected in 20% sucrose in PBS, frozen in O.C.T. compound with nitrogen liquid, and stored at -20°C until processing. Serial sections of frozen DRGs were cut in a cryostat (16 µm), mounted onto silanized slides, and used in immunohistochemical detection of individual PKC isoforms. Sections were incubated overnight at 4°C with one of the affinity-purified rabbit polyclonal antibodies listed below. Antibodies were diluted in PBS with 0.01% Triton X-100 (PBS-TX) to enhance penetration of antiserum into the tissue. The antibodies were anti-PKC (1:400), anti-PKCßI (1:250), anti-PKCßII (1:250), and anti-PKC
(1:250) (Santa Cruz). After brief rinses in PBS and 1-h incubation in diluted biotinylated goat anti-rabbit IgG, the sections were incubated for 1 h with Vectastain Elite ABC Reagent (Vector Laboratories). The antigen-antibody complexes were visualized by incubation in 0.1% 3,3'-diaminobenzidine in 0.1 mol/l PBS (pH 7.4) containing 0.001% H2O2. Sections were dehydrated through ascending concentrations of ethanol and coverslipped with Entellan (Merck).
Statistical analysis.
All data are expressed as means ± SE. Treatment effects were analyzed by one-way or two-way ANOVA with post hoc analysis by the Scheffé test. P < 0.05 was considered statistically significant.
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RESULTS |
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Immunoblotting of PKCs, nNOS, and SNS proteins.
By immunoblotting, the expression of PKCßII, nNOS, and SNS/PN3 proteins was significantly decreased by 39, 64, and 90% in DRGs of diabetic rats compared with control DRGs (Fig. 3). Other isozymes were also decreased in diabetic rats: PKC, 26%; PKCßI, 34%; and PKC
, 43%. TTX-R Na channel protein SNS2/NaN was not detected in all groups of DRGs. Treatment with LY333531 did not change the expression of proteins
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DISCUSSION |
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The mechanism of diabetic hyperalgesia remains unknown. Although STZ-diabetic animals show cachexia, malnourished condition, and ketosis from the uncontrolled hyperglycemia, there is no report that each of these conditions per se relates to hyperalgesia. However, the irritability due to general ill-health in STZ-induced diabetic animals may partly contribute to higher response to nociceptive stimuli (25). Aside from the latter point, many researchers have noticed the presence of mechanical hyperalgesia in STZ-induced diabetic animals. In fact, high glucose per se (26), changes of neurotransmitters (27), alterations of opioid metabolism and receptors (28, 29), or physiologically increased responsiveness or abnormalities of ion channels of neurons (15,18,30, 31,32) have been proposed as contributing factors to hyperalgesia. Many reports have supported the significant effect of PKC modulators on the generation of pain; PKC may contribute to primary afferent C-fiber excitability, because phorbol esters can depolarize cultured DRG neurons with C-fiber properties (33). Primary cultured DRG neurons in STZ-induced diabetic rats depolarize because of altered TTX-R Na+ channel activity (18). Ahlgren and Levine (15) reported the intradermal effect of PKC inhibitors on the reduced mechanical nociceptive threshold and on the increased C-fiber excitability in STZ-induced diabetic rats. The relief by intradermal injections of PKC inhibitors is consistent with our observation of the effect of LY333531 on neurons or nerve fibers.
Spinal and supraspinal contributions to hyperalgesia have important roles by acting at a spinal N-methyl-D-aspartate (NMDA) receptor (34). The induction of neuropathic pain by STZ-induced diabetic rats renders spinal cord opioid systems ineffective in producing anti-nociception for noxious heat, electrical, and pressure stimuli (29). The mechanisms proposed for this opioid resistance include downregulation or destruction of opioid receptors. This is probably mediated by increased production of protein kinase C following activation of NMDA receptors in postsynaptic cells (35,36). In addition, Igwe and Chronwall (14) provided evidence for inflammation-induced upregulation of membranous PKCßII activity of the lumbar spinal cord ipsilateral to the inflammation. This indicates activity-dependent alterations in the regulation of translocation and activation of PKCßII, and their involvement in the initiation and maintenance of hyperalgesia. Confirmation of this was obtained by quantitative immunohistochemical analyses, time-course for increases in the intensity of PKCßII immunoreactivity as well as in the activity of membrane-associated PKCßII paralleled inflammation-mediated changes in paw withdrawal latency and paw diameter. This observation could support that the PKCß-selective inhibitor LY333531 may have a significant effect on neurons at the spinal level.
In the peripheral nervous system, the localization of PKC isoforms has not been well examined in DRG neurons. We found that PKCßI and PKCßII were respectively localized in satellite cells and neurons, especially small neurons, of DRG, the latter of whose axons, afferent A- and C-fibers, conduct nociception. Together with the observation that both DRG neurons and peripheral nerves are directly modulated by PKC, these findings may be compatible with the notion that LY333531 would have a beneficial effect on hyperalgesia by directly affecting the nociceptive threshold at the peripheral nerve level. With respect to the activity of PKC and the presence of its isoforms in diabetic nerves, it remains controversial whether PKC activity in diabetic nerves is reduced (37,38), unchanged (7,39), or increased (40), and it also remains unclear which isoform is altered in diabetic nerves. Our results that the ratio of membrane to cytosol PKCßII activity was significantly higher in diabetic than in control nerves, and that LY333531significantly reduced phosphorylated PKCßII, may support increased PKCß II activity of DRG neurons and the inhibitory effect of LY333531 on PKCßII activity of DRG neurons in diabetes.
LY333531 significantly restored the decrease in cGMP content of DRGs under ex vivo as well as in vivo conditions, which has been consistently observed in diabetic rats. Together with our observation that the treatment with L-arginine improves the cGMP content of DRG as well as hyperalgesia in diabetic rats, decreased cGMP content may underlie diabetic hyperalgesia, and amelioration of cGMP content would contribute to relief of hyperalgesia. An antinociceptive effect of L-arginine in diabetic mice has also been reported by others (41). We have previously reported the possible implication of decreased cGMP content in DRGs, as well as the decrease in nNOS expression, in the genesis of pain in diabetic rats (14). NO is a highly reactive, rapidly diffusible gas synthesized from L-arginine by tissue- and cell-specific NOS. The calcium-calmodulin-dependent constitutive nNOS produces a low level of NO, which specifically interacts with and activates heme-containing soluble guanylyl cyclase in neighboring neuronal cells in a paracrine fashion. The signal is transduced via cGMP and cGMP-dependent protein kinases (PKG). The calcium current in chick embryo DRG neuron is suppressed by NO donors and membrane-permeable cGMP analog, (42) and the calcium channel is a substrate of PKG (43). Because calcium current is closely associated with nociception (44), decreased activity of the nNOS-cGMP pathway may be involved in the genesis of hyperalgesia. In fact, the cGMP-PKG pathway was shown not only to trigger some forms of persistent pain (45), but also to be critical for the induction of long-term sensitization of nociceptive sensory neurons (46).
Although the mechanism by which LY333531 ameliorates the decrease in cGMP content remains unclear, the interrelationship between PKC and the NO-cGMP pathway has been demonstrated in the diabetic state. An impairment of NO-dependent cGMP generation in glomeruli from diabetic rats is mediated in part by an activation of PKC (47). In SH-SY5Y human neuroblastoma cells, impaired glucose-mediated NO-dependent cGMP production was corrected by PKC agonists and reproduced by PKC inhibition (48), a finding that is just opposite to ours in the contributory role of PKC for NO-cGMP metabolism.
We previously reported that TTX-R Na current was shown to be increased in DRGs of diabetic rats (18). TTX-R Na channels play an important role in nociception (19), and the inhibition of PKC activity was shown to increase nociceptive threshold in diabetic rats (15). One might speculate that the effect of LY333531 may be mediated by inhibition of the TTX-R Na current through blockage of the phosphorylation of TTX-R Na channels. An alternate explanation may include an increase in the expression of TTX-R Na channels in diabetes. Our data exclude the latter possibility. TTX-R Na channel protein of DRGs was significantly decreased in diabetic rats compared with control rats, without any effect of LY333531 on their expression. Decreased TTX-R Na channel expression in diabetic state was also reported by others (49).
In conclusion, it was clearly demonstrated that the inhibition of PKCßII activity by LY333531 ameliorated hyperalgesia in diabetic rats. Although its precise mechanism remains unclear, a restoration of cGMP content in DRG neurons, through inhibition of PKCßII activity, may contribute, at least partially, to the amelioration of hyperalgesia. Further investigations are required to clarify the mechanisms of action of LY333531 as well as the pathogenic mechanisms of diabetic hyperalgesia. However, the significant effect of LY333531 on diabetic hyperalgesia appears to indicate the potential of LY333531 as a therapeutic compound for the pain syndrome in diabetes, as well as its usefulness as a research tool for diabetic hyperalgesia.
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ACKNOWLEDGMENTS |
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The authors are indebted to David J. Chang, Research Strategy Manager, Neurobiology Unit, Roche Bioscience, for the kind supply of anti TTX-R Na channels. The authors thank Dr. Louis "Skip" Vignati, Research Fellow, Lilly Research Laboratories, for his review of the manuscript.
Address correspondence and reprint requests to Hitoshi Yasuda, PhD, Division of Neurology, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. E-mail: hyasuda{at}belle.shiga-med.ac.jp
Received for publication August 20, 2002 and accepted in revised form May 1, 2003
CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; PKC, protein kinase C; PKG, cGMP and cGMP-dependent protein kinases; RIPA, radioimmunoprecipitation assay; STZ, streptozotocin; TTX, tetrodotoxin; TTX-R, TTX-resistant
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REFERENCES |
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