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Receptors and channels
Abstracts
11
(604) Decrease of TTX-resistant sodium current density in dorsal root ganglion neurons after hind paw incision
(606) Functional characterization of human ASIC3 expressed in Xenopus Oocytes and Cos-7 cells
C. Dalle, J. Eisenach; Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, NC Similar to nerve injury, surgical tissues injury has been shown to result in spinal, as well as, peripheral sensitization. Activation of C-fibers and increased spontaneous activity in A-beta fibers by innocuous stimulus occurs after hind paw incision (Zhan et al., 2002) and the mechanical response threshold is reduced in A-delta fibers and responsiveness to punctate mechanical stimuli is greater relative to control rat (Pogatzki et al., 2002). It has recently become clear that spontaneous firing and increase in excitability of primary afferent fibers involve variation in expression and/or redistribution of sodium channels. The aim of this study is to investigate the expression of sodium channel on DRG neurons after plantar hind paw incision (Brennan et al., 1996). For this purpose, sodium currents were recorded in the whole-cell patch-clamp configuration on freshly isolated DRG neurons from naı¨ve rat (no surgery) and from rat with hind paw incision. All rat were injected with Fluorogold solution in the plantar surface of the hind paw to identify the somata of DRG neurons with fibers innervating the surgery wound. Mechanical allodynia was assessed by determining of the paw withdrawal threshold by application of calibrated von Frey filaments. Fluorogold caused any measurable changes in the behavior of either naive or postoperative animals. Only rats with a significant mechanical allodynia 2 days following surgery were further included for study. Our result show that hind paw incision is associated with decrease of TTX-resistant sodium current density in small and large DRG neurons. No significant variation of TTXsensitive sodium current density was found. It is difficult to predict how this reduction in TTX-resistant current density might impact excitability, but we can hypothesize for a redistribution of sodium channel in axons, which may contribute to the greater spontaneous firing and increased excitability seen after surgical tissues injury.
A. Zou, D. Printzenhoff, Z. Lin, P. Miu, A. Wickenden; Icagen, Inc., Durham, NC Six members of the ASIC sub-family have been cloned to date. Most ASIC channels are highly expressed in sensory neurons and sympathetic cardiac afferents, where they are thought to play an important role in peripheral pain perception and mechanotransduction. While the properties of rat ASIC channels have been studied in detail, the functional properties of human orthologs have not been studied extensively. The purpose of the present study was to characterize the functional properties of human ASIC3 expressed in Xenopus oocytes and Cos-7 cells. In both expression systems, hASIC3 currents were activated by changing the pH of the bathing solution and exhibited rapid desensitization on exposure to acidic solutions. Channel activation was steeply pH dependent, with half maximal current activation occurring at a pH of 6.4 (Cos-7) and 6.4 (oocytes), each with a slope value of 29 and 30 respectively. In Cos-7 cells the ASIC3 I-V relationship was linear over a wide voltage range and the current reversal potential was ⫹45 mV. hASIC3 channels exhibited sub-threshold desensitization, with channel availability being decreased when the pH of the bathing solution was ⬍8. Channel availability was reduced by approximately 50% at physiological pH. hASIC3 currents were inhibited by amiloride with IC50 values of 19 M and 31 M in Cos-7 cells and Xenopus oocytes, respectively. Large diameter rat DRG neurons exhibit native acid sensitive currents comprising both transient and sustained components. The pH0.5 for the transient component was very similar to that of ASIC3 (6.3, slope ⫽ 29) suggesting a possible role for this channel in native DRG acid-sensitive currents. The steep pH dependence of ASIC channels in cloned ASIC3 cells and in DRG neurons suggests that these channels can function as a rapid on/off switch in response to subtle changes to extracellular pH under normal and pathological conditions.
(605) Analyses of Inter-Individual differences in Nociception and Morphine Analgesia in relationship to A118g MuReceptor polymorphism
(607) Effects of AMPA receptor activity on exploratory behavior in a rat visceral pain model
M. Van Dijk, S. Simons, M. van der Werf, R. Van Lingen, J. Van den Anker, D. Tibboel, R. van Schaik; Erasmus MC, Rotterdam Effective morphine requirements during neonatal intensive care treatment are difficult to predict as neonatal infants show large inter-individual variability in nociception and pain response after morphine administration. The objective was to determine the effects of the A118G single nucleotide polymorphism (SNP) of the ı`-receptor gene on the inter-individual variability of nociception and morphine requirements among neonatal patients. The DNA of 118 neonates, who participated in a randomized placebo controlled trial investigating the effects of continuous morphine in ventilated neonates, was analyzed for the A118G SNP. (Simons SH et al, JAMA 2003) Relationships between the different genotypes and pain scores and morphine requirements were investigated. Eighty-four patients were wild type, 30 patients were heterozygous and 4 patients were homozygous for the mutation. No significant differences in pain scores and morphine requirements were found between the wild type and heterozygous patients. No homozygous patient needed additional morphine. No relationship between the ı`-receptor A118G genotype and inter-individual variability in pain scores or morphine requirements was detected in our study population. In other words genotype analysis does not preclude morphine need.
G. Su, L. Ma, J. Wu, X. Zhang, Q. Lin, W. Willis, L. Fang; University of Texas Medical Branch, Galveston, TX AMPA receptors are one type of important ionotropic glutamate receptors in the central nervous system and have been reported to mediate nociceptive transmission in the spinal cord. The current project was designed to study the role of spinal AMPA receptors in animals following visceral noxious stimuli. Visceral noxious stimuli were induced in rats by intracolonic injection of mustard oil. NBQX, an antagonist of AMPA receptors, was administrated through a pre-implanted intrathecal catheter before visceral stimulation. Exploratory behavior activity was examined to observe the changes in exploratory behavior in an open-field box equipped with monitors for detecting activity. Results showed that visceral nociception induced by intracolonic injection of mustard oil significantly reduced exploratory behavior in rats, including decreased traveled distance, entry time, total activity, rearing time, rearing activities and increased resting time. However, NBQX significantly reversed these visceral noxious stimulation-induced behavioral changes. In addition, the effect of NBQX on the expression of a transcription factor, cAMP-responsive element-binding protein (CREB) and phosphorylated CREB, in the lumbosacral spinal cord of rats was detected by using quantitative Western blots. We found that intra-colonic application of mustard oil significantly increased the phosphorylation of CREB in the lumbosacral spinal cord and pre-treatment with NBQX significantly blocked the increase in expression of phosphorylated CREB protein. However, there was no change in the expression of CREB protein in each group. These results suggest that AMPA receptors play an important role in mediating central visceral nociceptive transmission, which might involve regulation of the phosphorylation of the transcription factor, CREB. Supported by NIH NS09743, NS11255, DK 56338 F/P Grant and John Sealy Fund Grant 2591-02.
11
(604) Decrease of TTX-resistant sodium current density in dorsal root ganglion neurons after hind paw incision
(606) Functional characterization of human ASIC3 expressed in Xenopus Oocytes and Cos-7 cells
C. Dalle, J. Eisenach; Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, NC Similar to nerve injury, surgical tissues injury has been shown to result in spinal, as well as, peripheral sensitization. Activation of C-fibers and increased spontaneous activity in A-beta fibers by innocuous stimulus occurs after hind paw incision (Zhan et al., 2002) and the mechanical response threshold is reduced in A-delta fibers and responsiveness to punctate mechanical stimuli is greater relative to control rat (Pogatzki et al., 2002). It has recently become clear that spontaneous firing and increase in excitability of primary afferent fibers involve variation in expression and/or redistribution of sodium channels. The aim of this study is to investigate the expression of sodium channel on DRG neurons after plantar hind paw incision (Brennan et al., 1996). For this purpose, sodium currents were recorded in the whole-cell patch-clamp configuration on freshly isolated DRG neurons from naı¨ve rat (no surgery) and from rat with hind paw incision. All rat were injected with Fluorogold solution in the plantar surface of the hind paw to identify the somata of DRG neurons with fibers innervating the surgery wound. Mechanical allodynia was assessed by determining of the paw withdrawal threshold by application of calibrated von Frey filaments. Fluorogold caused any measurable changes in the behavior of either naive or postoperative animals. Only rats with a significant mechanical allodynia 2 days following surgery were further included for study. Our result show that hind paw incision is associated with decrease of TTX-resistant sodium current density in small and large DRG neurons. No significant variation of TTXsensitive sodium current density was found. It is difficult to predict how this reduction in TTX-resistant current density might impact excitability, but we can hypothesize for a redistribution of sodium channel in axons, which may contribute to the greater spontaneous firing and increased excitability seen after surgical tissues injury.
A. Zou, D. Printzenhoff, Z. Lin, P. Miu, A. Wickenden; Icagen, Inc., Durham, NC Six members of the ASIC sub-family have been cloned to date. Most ASIC channels are highly expressed in sensory neurons and sympathetic cardiac afferents, where they are thought to play an important role in peripheral pain perception and mechanotransduction. While the properties of rat ASIC channels have been studied in detail, the functional properties of human orthologs have not been studied extensively. The purpose of the present study was to characterize the functional properties of human ASIC3 expressed in Xenopus oocytes and Cos-7 cells. In both expression systems, hASIC3 currents were activated by changing the pH of the bathing solution and exhibited rapid desensitization on exposure to acidic solutions. Channel activation was steeply pH dependent, with half maximal current activation occurring at a pH of 6.4 (Cos-7) and 6.4 (oocytes), each with a slope value of 29 and 30 respectively. In Cos-7 cells the ASIC3 I-V relationship was linear over a wide voltage range and the current reversal potential was ⫹45 mV. hASIC3 channels exhibited sub-threshold desensitization, with channel availability being decreased when the pH of the bathing solution was ⬍8. Channel availability was reduced by approximately 50% at physiological pH. hASIC3 currents were inhibited by amiloride with IC50 values of 19 M and 31 M in Cos-7 cells and Xenopus oocytes, respectively. Large diameter rat DRG neurons exhibit native acid sensitive currents comprising both transient and sustained components. The pH0.5 for the transient component was very similar to that of ASIC3 (6.3, slope ⫽ 29) suggesting a possible role for this channel in native DRG acid-sensitive currents. The steep pH dependence of ASIC channels in cloned ASIC3 cells and in DRG neurons suggests that these channels can function as a rapid on/off switch in response to subtle changes to extracellular pH under normal and pathological conditions.
(605) Analyses of Inter-Individual differences in Nociception and Morphine Analgesia in relationship to A118g MuReceptor polymorphism
(607) Effects of AMPA receptor activity on exploratory behavior in a rat visceral pain model
M. Van Dijk, S. Simons, M. van der Werf, R. Van Lingen, J. Van den Anker, D. Tibboel, R. van Schaik; Erasmus MC, Rotterdam Effective morphine requirements during neonatal intensive care treatment are difficult to predict as neonatal infants show large inter-individual variability in nociception and pain response after morphine administration. The objective was to determine the effects of the A118G single nucleotide polymorphism (SNP) of the ı`-receptor gene on the inter-individual variability of nociception and morphine requirements among neonatal patients. The DNA of 118 neonates, who participated in a randomized placebo controlled trial investigating the effects of continuous morphine in ventilated neonates, was analyzed for the A118G SNP. (Simons SH et al, JAMA 2003) Relationships between the different genotypes and pain scores and morphine requirements were investigated. Eighty-four patients were wild type, 30 patients were heterozygous and 4 patients were homozygous for the mutation. No significant differences in pain scores and morphine requirements were found between the wild type and heterozygous patients. No homozygous patient needed additional morphine. No relationship between the ı`-receptor A118G genotype and inter-individual variability in pain scores or morphine requirements was detected in our study population. In other words genotype analysis does not preclude morphine need.
G. Su, L. Ma, J. Wu, X. Zhang, Q. Lin, W. Willis, L. Fang; University of Texas Medical Branch, Galveston, TX AMPA receptors are one type of important ionotropic glutamate receptors in the central nervous system and have been reported to mediate nociceptive transmission in the spinal cord. The current project was designed to study the role of spinal AMPA receptors in animals following visceral noxious stimuli. Visceral noxious stimuli were induced in rats by intracolonic injection of mustard oil. NBQX, an antagonist of AMPA receptors, was administrated through a pre-implanted intrathecal catheter before visceral stimulation. Exploratory behavior activity was examined to observe the changes in exploratory behavior in an open-field box equipped with monitors for detecting activity. Results showed that visceral nociception induced by intracolonic injection of mustard oil significantly reduced exploratory behavior in rats, including decreased traveled distance, entry time, total activity, rearing time, rearing activities and increased resting time. However, NBQX significantly reversed these visceral noxious stimulation-induced behavioral changes. In addition, the effect of NBQX on the expression of a transcription factor, cAMP-responsive element-binding protein (CREB) and phosphorylated CREB, in the lumbosacral spinal cord of rats was detected by using quantitative Western blots. We found that intra-colonic application of mustard oil significantly increased the phosphorylation of CREB in the lumbosacral spinal cord and pre-treatment with NBQX significantly blocked the increase in expression of phosphorylated CREB protein. However, there was no change in the expression of CREB protein in each group. These results suggest that AMPA receptors play an important role in mediating central visceral nociceptive transmission, which might involve regulation of the phosphorylation of the transcription factor, CREB. Supported by NIH NS09743, NS11255, DK 56338 F/P Grant and John Sealy Fund Grant 2591-02.