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Bradykinin and Nerve Growth Factor (NGF) Lowered the Heat-Activation Threshold of Capsaicin Receptor (VR1) in Primary Nociceptive Neurons - Mechanisms of Heat-Hyperalgesia in Inflamed Condition
Takeshi Sugiura, M.D.; Hirotada Katsuya, M.D., Ph.D.; Kazue Mizumura, M.D., Ph.D.
Anesthesiology and Resuscitology, Nagoya City Univ. Med. Sch., Nagoya, Aichi, Japan
BACKGROUND: Hyperalgesia to heat is commonly observed in inflamed tissues, and inflammatory mediators are considered to sensitize nociceptors. Capsaicin receptor, VR1, is a sensory neuron-specific ion channel that is activated by capsaicin, acidification and noxious heat (1, 2). We hypothesized that one of the mechanisms for nociceptor sensitization to heat is the modulation of VR1 thermal activity by inflammatory mediators, and demonstrated that the heat activation threshold of VR1 was dramatically lowered down to the physiological temperature through bradykinin receptor (B2R) expressed in heterologous system (3). In this study, we investigated whether the threshold temperature of heat-activated currents that are assumed to be mediated through VR1, are also reduced by inflammatory mediators in primary sensory neurons from intact and persistently inflamed rats.

METHODS: Male Wistar rats were given interdermal injections of a suspension of complete Freund's adjuvant (1.0 mg/kg) into the tail under ether anesthesia. Two weeks after inoculation, the rats that showed a swelling of the hind paw were used for the experiment. Untreated rats were used as the control. The dorsal root ganglia (DRGs) were isolated and treated with collagenase, then the DRG neurons were mechanically dissociated on poly-L-lysine-coated glass coverslips and were kept in DMEM with NGF at 37°C for 2 days. For thermal stimulation, bath temperature was increased at a rate of about 1.0°C/sec using a pre-heated extracellular solution. The heat-evoked currents in cultured DRG neurons were examined by means of whole-cell patch-clamp recordings. This study was approved by the Animal Care Committee of the Nagoya University. Statistical analysis of the data was done by unpaired t -test or one way ANOVA. p < 0.05 was considered as statistically significant.

RESULTS: Heat-evoked currents developed at 42.0±0.5°C (n=5,control) with a steep temperature dependency in capsaicin-sensitive neurons obtained from intact rats. Bradykinin (10 and 100 nM) pre-treatment significantly lowered the threshold temperature (32.2±2.4°C; n=6, 24.0±1.0°C; n=5, respectively, p < 0.05, vs. control group). Moreover the effect of bradykinin was abolished by a protein kinase C inhibitor, calphostin C, suggesting that the thermal threshold of VR1 activity was modulated by phosphorylation of this ion channel. While in DRG neurons from inflamed rats, the heat threshold temperature was reduced in the absence of any exogenous irritants (26.2±2.2°C, n=6, p < 0.05). This lowered-threshold temperature returned to that of DRG neurons from intact rats by treating them with anti-NGF antibody for more than 90 min before recording (42.3±1. 4°C; n=5).

CONCLUSIONS: We conclude that bradykinin and NGF might be potent substances that sensitize VR1 to lower its thermal activation threshold in acute or chronic inflammation model. Present results suggest that the lowering of the heat threshold temperature of VR1 activation might be a peripheral mechanism of thermal hyperalgesia as well as spontaneous pain in inflammation.

REFERENCES: (1) Nature 389, 816-824. (1997), (2) Neuron 21, 531-543. (1998), (3) Soc. Neurosci. Abst. 27: Program No. 925.2 (2001)

Anesthesiology 2002; 96: A949