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Receptors and channels




Tom Meuser. Anja Gabriel*, Christian Pietruck, Guo-xi Xie*, Stefan Grond, Pamela Pierce Palmer, Depts. of Anesthesia, Univ of California San Francisco, USA and Univ of Cologne, 50924 Koln. Germany

Aim of Investigation: Pharmacoiogical studies have been proposed that selective inhibition of the 5-HT4 receptor subtype reduces inflammatory pain and mediates antinociccption in enteric viscera and in cutaneous terminals. The distribution of 5-HT4 receptors and their role in the central nervous tissue were studied by binding assays and in situ hybridization, however, no previous study has attempted to resolve the existence of5-HT4 receptor subtype mRNA in rat peripheral nervous tissue. Aim of this investigation was to determine the presence of5-HT4 mRNA in lumbar dorsal root ganglia (DRG), superior cervical ganglia (SCO) and lumbar sympathetic ganglia (LSG) using the method ofpolymerase chain reaction (PCR).

Methods: After institutional approval, DRGs, SCGs, LSGs and hippocampal formation (positive control) were dissected from 3 anesthetized male Sprague Dawley rats and immediately frozen in liquid nitrogen. Oligonucleotide primers (5'-3': 807-826; 3'-5':990-971) were chosen based on unique regions of complimentary DNA sequence for the rat 5-HT4 receptor subtype framing a 184 bp sequence. After reverse transcription, PCR amplification was performed and the resulting cDNA products were separated on aga-rose gels using electrophoresis. The cDNA products were then subcloned and sequenced to prove receptor subtype identity.

Results: The presence of 5-HT4 receptor subtype mRNA was clearly detected in DRGs, SCGs and hippocampal formation, however, no 5-HT4 mRNA could be detected in LSGs.

Conclusions: The cloned rat 5-HT4 receptor is present in DRG and SCG, underlining the pharmacological studies that the 5-HT4 receptor subtype is essential when analyzing peripheral 5-HT actions on sensory afferents and sympathetic efferents, such as the production of pain and hyperalgcsia. Findings from this study may direct further efforts to determine the role ofserotonin receptors in the peripheral nervous system.


Catherine Abbadie. Y.-X. Pan* and G.W. Pasternak, The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Aim of Investigation: We have identified new splice variants of the mouse mu-opioid receptor (MOR-1) gene, whose fourth exon undergoes alternative splicing. The aim of the present study was to compare the distribution ofMORl with the distribution of the three splice variants, MOR-1 C, MOR-1D and MOR-1E using immuno-histochemistry.

Methods: Anti-peptide anti-sera to the C-terminal peptide sequence from the new fourth exons were raised in rabbit (Multiple Peptide Systems). The specificity of the antibodies was determined using transfected CHO cells, Western blots and immunoisolation studies.

Results: The relative abundance of MOR-1-like immunoreactivny (-LI) as compared to MOR-1 C-LI can be categorized in three ways. (1) The first group includes regions in which MOR-1-LI was the predominant form expressed. These areas include the striatum, hippocampal formation, prcsubiculum and parasubiculum, amyg-daloid nuclei, thalamic nuclei, locus coeruleus and ambiguus nucleus. (2) In a few CNS regions including the lateral parabrachial nucleus and laminae 1-11 of the spinal trigcminal nuclei and the spinal cord, MOR-1 C and MOR-1-LI were both prominent. (3) The final group contained greater MOR-1 C-LI than MOR-1-LI in the lateral septum, most hypothalamic nuclei such as the median eminence, pcrivcntricular, suprachiasmatic, supraoptic, arcuate, para-ventricular, vcntromcdial and dorsomedial nuclei, and the deep laminae of the spinal cord. Double labeling studies showed colo-calization of the two receptors in neurons of the lateral septum, and in a few processes in the substantia gclatmosa of the spinal cord, but not in the median eminence or in the arcuate nucleus where both MOR are expressed. We arc currently looking at the localization of MOR-1D and MOR-1 E.

Conclusions: These differences in regional distribution show a different distribution from a common gene. It remains to be determined the relative contribution ofMORIC and MOR1 to different functions. Acknowledgments: Supported by NTDA DA 02615.


U. Stamer*. Q. Shu*, A. Hocft*, F. Stuber* (SPON: C. Dierke-Dzierzon), Dept ofAnaesthesiology and Intensive Care Medicine, Univ of Bonn, 53105 Bonn, Germany

Aim of Investigation: Tolerance to opioids may be caused by modulation ofopioid receptor expression. This study investigates the influence ofsufcntanil, a potent ^i-opioid receptor agonist, on opioid receptor expression in SH-SY5Y cells.

Methods: After approval of the local ethics committee a human neuroblastoma cell line (SH-SY5Y) was cultured at 37C and 5% CO; together with 0, 0.1 and 10.0 ng/ml sufentanil for one to six weeks. After RNA isolation and cDNA synthesis, reverse tran-scriptase polymerase chain reaction was performed. PCR products were analysed by agarose gel electrophoresis. RNA contents were estimated by scmiquantitative analysis employing a housekeeping gene (GAPDH) as internal standard. Southern blot analysis and hybridization to a specific DIG-labcled probe were performed to confirm the identity of^- and 5-receptor transcripts.

Results: In all samples 4- and 5-opioid-receptors were expressed constitutivcly. Semiquantitative PCR analysis of amplified cDNA revealed that the expression of both receptors was not influenced by incubation with different concentrations of the potent u-receptor agonist sufentanil for 1, 2, 3, 4, 5, and 6 weeks. A downrcgulation of^i- and 6-opioid-reccptor expression was not observed in this long-term culture with sufentanil. All amplified PCR products proved their specifity for \x- and 6-opioid-reccptors by hybridization to the specific probes.

Conclusions: The expression of [i- and 5-opioid receptor gene sequences in SH-SY5Y cells was not influenced by incubation with sufentanil. The development of symptoms like tolerance and addiction in patients treated by opioids seems not to be modulated on the level of receptor expression.


Daniela Oddiah*. Martin Koltzenburg, Dept of Neurology, Univ of Wurzburg, Josef-Schneider-Str. 11, D-97080, Wurzburg, Germany.

Aim of Investigation: To characterise the expression of the voltage-gated sodium channel a-subunit NaN, which partially mediates the TTX-resistant (TTX-R) sodium current in small dorsal root ganglion (DRG) neurons usually classified as nociceptors.

Methods: Adult mouse DRG neurons were cultured for 24-48 hours without additional trophic factors. Small diameter cells were classified by their ability to bind the lectin 1B4 that provides a vital stain ofnon-peptidergic nociceptive neurons. Individual 1B4 positive or negative cells were harvested using glass micropipettes and mRNA was reverse transcribed using oligo (dT) primers. The house-keeping gene, p-actin, was used as a positive control of harvested cells which were then investigated for sodium channel gene expression.

Results: Of 39 cells from which actin could be amplified, no 1B4-positive cells of any size expressed NaN mRNA (n = 18, mean diameter = 21.3 3.6um). NaN mRNA was however associated with a wide range of cell sizes in IB4-negative cells (n = 21, mean diameter = 24.6 8.6um). Both small cells of<30u.m diameter classified as nociceptors, as well as larger cells, up to 40um, showed a robust expression of NaN mRNA. Conclusions: Single cell RT-PCR can be used to characterise sodium channel gene expression in DRG neurons in association with classical biochemical markers. The present results illustrate that NaN expression occurs in a wide size range of DRG neurons but appears to be restricted to 1B4 negative, peptidergic neurons.


V.H. John*. Z.M. Gladwell, D.J. Trezise*, C.A. Hick, D.T. Grose*, S.N. Tate & X.M. Xie * (SPON: C. Duffy), Neuroscience unit, Glaxo Wellcome R&D, Stevenage, Hens, SG1 2NY, UK

Aim of Investigation: Two novel, voltage-gated tetrodotoxin resistant (TTXiO sensory neuron-specific sodium channel cx-subunits, named SNS/PN3 and SNS2/NaN have recently been cloned from rat dorsal root ganglia (DRG). Here, we have investigated the functional properties of these two channels when heterologously expressed in HEK293T cells and compared these properties to those of the native TTXn current in rat sensory neurones,

Methods: Whole cell patch clamp recordings were made from tran-siently transfected HEK293T cells and acutely dissociated small diameter rat DRG neurones (10-25u.m). A range ofbiophysical properties were determined using different voltage command protocols.

Results: Transiently expressed SNS/PN3 and SNS2/NaN Na" channels conduct ttxr Na* currents. Concentration-response curves to TTX gave half-inhibitory concentrations of 31 u.m and luM for SNS/PN3 and SNS2/NaN, respectively. SNS2/NaN currents were activated at much more negative potentials than SNS/PN3 (Vi/2 values of activation were - 452mV and +l3mV, respectively). Kinetic studies revealed that although both channel types had similar rates of activation, SNS2/NaN inactivated considerably faster than SNS/PN3 (t values from peak current were 1.20.1ms and 11.62.3ms, respectively). Recordings made from DRG neurones indicate that TTXRis a heterogeneous population of voltage-gated Na+ channels that encompass both SNS/PN3 and SNS2/NaN biophysical properties.

Conclusions: When transiently expressed in HEK293T cells, SNS/PN3 and SNS2/NaN encoded TTX-insensitive channels with some of the functional characteristics of native TTXp. The heterogeneous properties ofTTXR in sensory neurones may reflect different expression patterns of SNS/PN3 and SNS2/NaN.


Amir R*. Michaelis M.,Devor M, Dept. of Cell and Animal Biology, Life Sciences Inst., Hebrew Univ. of Jerusalem, Jerusalem 91904, ISRAEL

Aim of Investigation: To investigate the mechanism of the abnormal (ectopic) discharge that develops in many primary sensory neurons following peripheral nerve injury. This repetitive discharge has been shown to contribute to chronic pain states.

Methods: Intracellular recording both from non-axotomized and chronically axotomized rat dorsal root ganglion (DRG) neurons in vitro.

Results: A significant proportion of DRG neurons, both neurons with myelinated axons (A-neurons) and neurons with unmyelinated axons (C-neurons), exhibited subthreshold oscillations in their membrane potential. These oscillations were voltage sensitive as their prevalence, amplitude and frequency were all increased upon depolarization. The oscillations appear to be an essential substrate for repetitive ectopic discharge as they trigger spike activity. Only neurons with oscillations fired repetitively on depolarization. Cells without oscillations never fired repetitively even when deeply de-polarized (n=165). Nerve injury increased the prevalence of neurons with subthreshold oscillations (17/120 oscillating non-injured neurons, vs. 34/95 oscillating injured neurons; p<0.0005 chi-square test) and correspondingly the prevalence of the ectopic discharge. The oscillations depend on Na^ conductance. Lowering the concentration ofNa in the superfusing solution selectively eliminated the oscillations and the resulting ectopic discharge without blocking spike propagation (n=5/5 cells). Similar results were obtained using low concentrations of either tetrodotoxin (TTX) or lidocaine (n=l 3/13 cells).

Conclusions: Sodium-dependent membrane oscillations trigger the ectopic discharge in DRG neurons. Pharmacological suppression of the oscillations may provide a practical approach to the management of currently intractable chronic pain states, many of which are caused by ectopic afferent discharge.

Acknowledgments: Supported by grants from the BSF and the GIF. M. Michaelis was supported by DFG Grant Mi 457/2-1.


Jin-ichi Kobayashi* '. Masahiro Ohta*, Yoshihiro, Terada*', (SPON: M. Muraoka), Dept of 'Prosthetic Dentistry I and Physiology, Fac of Dentistry, Kyushu Univ, 3-1-1 Maidashi, Hi-gashi-ku, Fukuoka 812-8582, Japan

Aim of Investigation: Immunohistochcmical studies have revealed the existence of subgroups of the peripheral C-fibers, providing evidences for differential co-localization ofneuropeptides in their subgroups. To our knowledge little has been, however, studied yet for the heterogeneity ofC-fibers at the level of channels, nothing on the tetrodotoxin-resistant (TTX-R) C-fibers. Thus, we examined the heterogeneity ofelcctrophysiological properties in TTX-R C-fibers.

Methods: The existence of calcium-activated K^ channels (Kca) was investigated in fast and slow TTX-R C-fibers of the frog sciatic nerve by means of compound action potential recordings following repetitive and/or single stimulation. The time course of the fast and slow TTX-R C-fibers responses and the refractory period of them were measured.

Results: Post-tetanic positive potentials ofTTX-R C-fibers were partially inhibited after application of 100 nM charybdotoxin (CTX). The augmentation of the responses of slow TTX-R C-fibers to single pulse stimulation following repetitive stimulation was reduced after application of 100 nM CTX, while that of fast ones was little changed. The decay time constant of the augmentation of slow TTX-R C-fibers responses was reduced as well, while that of fast ones was not. Furthermore, the refractory period of slow TTX-R C-fibers was reduced after the blockade ofKca by 100 nM CTX, while that of fast ones was not.

Conclusions: The results suggest that TTX-R C-fibers have and heterogeneity for the distribution of in the peripheral nervous system, indicating the possibility that they might differentiate their functional roles in the peripheral processing of pain.


K. Dale, R. Brazdil, C. Hick, C. Kozlowski, S. Tate and C. Boun-tra, Neuroscience Unit, Glaxo Wellcome R&D Ltd., Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.

Aim of Investigation: Increased voltage-gated sodium channel (VGSC) activity in sensory neurones may contribute to the hyper-excitability of these neurones and the induction of central sensiti-sation in chronic pain states. Previous in si!u hybridisation and immunocytochemical studies have shown that expression of the TTX-R VGSCs SNS and SNS2 is confined to such sensory neurones. The aim of these experiments was to investigate alterations in expression ofmRNA for SNS and SNS2 in small (<600?m2), medium (600-1 lOO ?m 2) and large (>1 lOO ?m 2) diameter sensory neurones in a rat model of chronic inflammatory pain.

Methods: Male Random Hooded rats (200g) were injected (ipl, lOOul) with either Freunds Complete Adjuvant (FCA, Img/ml;n=12) or saline (n=12). Weight-bearing measurements were determined using a dual weight averager at 6h, 3d, 7d and 14d post FCA/saline. The animals (n=3 saline treated, n=3 FCA treated, per timepoint) were culled and lumbar dorsal root ganglia (L4-5) were removed. These tissues were sectioned (14p.m) and processed for visualisation of SNS and SNS2 mRNA. In saline treated control animals SNS mRNA was shown to be localised in 68% of small, 54% of medium and 29% of large diameter sensory neurone cell bodies. While SNS2 mRNA was shown to be localised in 65% of small, 34% of medium and 10% of large diameter sensory neurone cell bodies. In our rat model of chronic inflammatory pain, SNS1 mRNA expression appears to be down regulated in the small diameter neurone cell bodies at 3d post FCA but upregulated in the large diameter fibres at 7d post FCA compared to saline controls. In contrast SNS2 expression appears to be upregulated in small, medium and large diameter at 7 days post FCA compared to saline controls.

Conclusions: SNS and SNS2 mRNA expression appears to be dif-ferentially modulated in these sensory neurones following a peripheral inflammation. This study would suggest that alterations in sensory neurone specific sodium channel expression may contribute to abnormal processing ofnociceptive information in chronic pain states.


F. Porreca. J. Lai, M.H. Ossipov, S. Wegert*, D. Bian, 'S. Novakovich and 'J. C. Hunter, Depts of Pharmacology and Anesthesi1ology, Univ of Arizona Health Sciences Center, Tucson, AZ, 85724, USA and 'Center for Biological Research, Roche Bio-science, Palo Alto, CA 94304 USA.

Aim of Investigation: Increased spontaneous activity of peripheral nerves after injury is believed to be a driving force behind the establishment of central sensitization and behavioral signs ofneuro-pathic pain. The studies presented here were designed to test the importance of PN3/SNS, a TTX-R sodium channel, in the manifestations ofneuropathic pain from experimental diabetes or NGF.

Methods: Male SID rats were used in all experiments. Tactile allo-dynia was determined with von Frey filaments and thermal hyper-algesia by radiant heat applied to the hindpaw. Diabetic neuropathy was induced with a single injection of streptozotocin (STZ) (75 mg/kg, i.p.). Elevated blood glucose (>14 mM), tactile allodynia and thermal hyperalgesia occurred within 28 days. Neuropathic pain was also present for several days after a single injection of NGF (1 mg/kg, i.p.). Rats received antisense (AS) or mismatch (MM) ODN to PN3/SNS (45 ug, b.i.d. via catheters starting after the onset of diabetic related allodynia/hyperalgesia or 36 hrs prior to NGF injection. AS or MM injections were continued for up to 7 days.

Results: The injections of AS ODN to PN3/SNS blocked both tactile allodynia and thermal hyperalgesia in diabetic rats, whereas MM ODN or saline did not. Responses returned to the neuropathic state within 48 hrs of cessation of AS ODN administration. AS ODN did not alter responses in normal rats. Similarly, pretreatment with AS, but not MM, ODN blocked the development of tactile allodynia and thermal hyperalgesia after NGF. STZ or NGF- induced elevation of PN3/SNS was confirmed by immunohisto-chemistry. AS ODN "knocked-down" PN3/SNS expression below that of non-treated rats, whereas MM ODN had no effect on PN3/SNS levels in any group.

Conclusion: The results presented here extend the importance of the PN3/SNS channel to include the manifestation of signs ofneuropathic pain elicited by physiologic mechanisms, as in diabetic neuropathy or elevated expression of NGF.


K. Coward, G. Saldanha, R. Birch, T. Carlstedt, P. Anand. Dept of Neurology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd, London W12 ONN, UK

Aim of Investigation: To relate the presence of SNS1/PN3 and SNS2/NaN Na channels in human tissues to pain states.

Methods: Immunostaining of tissue sections with specific SNS1, 2 antibodies (from GlaxoWellcome).

Results: SNS1/PN3- and SNS2-like immunoreactivity (-IR) was seen in normal nerves, skin, and control postmortem dorsal root ganglia, predominantly in small sensory fibres. There was a parallel decrease of SNS1/PN3- and SNS2-IR in human sensory neu-ronal cell bodies and an increase in some injured nerve fibres acutely after brachial plexus injury, suggesting that pre-synthes-ized, intracellularly-located channel proteins may undergo translo-cation to peripheral nerves with accumulation at sites of injury, or that their expression is reduced after axotomy, or both. However, nerve terminals in target tissues and neuromas from patients with persistent pain, hyperalgesia, and allodynia showed marked increases ofSNSl/PN3-IR, but little or no SNS2-IR, suggesting that the former may be necessary for the allodynic phenotype. Immunoreactivity to both channels was similar to control nerves in the majority ofsural nerve trunk biopsies in a selection of human neu-ropathies, irrespective of nerve inflammation or demyelination.

Conclusions: SNS1/PN3 blocking agents are most likely to be effective in patients with chronic target organ injury/inflammation, and with nerve injury. Local hypersensitivity may be the best target for such treatment, and monitor of its efficacy.


'Dasa Cizkova, 'Martin Marsala, Ken Stauderman*, 'Tony Yaksh. 'Dept. ofAnesthesiology, Univ, of California San Diego, La Jolla, CA 92093, USA ^IBIA Neurosciences, Inc, La Jolla, CA 92037, USA

Aim of Investigation: Define the distribution ofCa2* channel alB subunit in spinal cord (SC) and dorsal root ganglion cells (DRGs) in rats before and at 1,2, 4, 7, 10 and 15 days after nerve injury.

Methods: The left L5, L6 spinal nerves were tightly ligated to induce tactile allodynia (threshold < 2.5-3 grams) as defined with Von Frey filaments. Cryostat sections of DRGs and SC (L3-S1 segments) from control and lesioned animals were processed for standard ABC peroxidase technique using a specific polyclonal antibody (pAb) to recognize the alB subunit of voltage-gated calcium channels.

Results: Tactile allodynia developed only in nerve ligated rats from 5 days post surgery. Immunostaining of control sections with alB pAb showed an evenly distributed staining pattern throughout all laminae of the dorsal hom, with a higher density seen on the soma ofa-motor neurons, and all DRG cell types. After nerve ligation, a significant increase in alB immunoreactivity was detected in i) SC: as punctate staining pattern in lamina II during 4-15 days post surgery (with unchanged staining pattern in the a-motoneurons); and ii) on the cell bodies of small DRG cells during short term (7D) survival and on all cell types during the longer (10-15D) postliga-tion period.

Conclusions: Following nerve injury, alB Ca2+ channel expression increases in the small DRG cells. This correlates with increased alB in SC lamina II, suggesting that N-type Ca2+ channels are increased on the terminals of small afferents that carry nociceptive information. These results may help account for the activity of N-type Ca2+ channel blockers in nerve injury pain states.

Acknowledgments: Supported in part by SIBIA Neurosciences, Inc and by NIH Grant NS 16541


Richard Lewis'. Denise Adams '', lain Sharpe "', Marion Lough-nan'', Trudy Bond'", Linda Thomas'', Alun Jones'', Jodi-Lea Matheson3', Roger Drinkwater', Katherine Nielsen'', David Craik'', Alan Robertson4', Paul Alewood'', 'Centre for Drug Design & Development, ^PI Agricultural Biotechnology Centre, ^SIRO Tropical Agriculture, of The Univ of Queensland, 4072, Australia, and "AMRAD Operations Pty Ltd, Richmond, 3121, Australia

Aim of Investigation: To discover new N-type selective co-conopeptides from Australian cone snails that may be useful in the control of pain.

Methods: Conopeptides were discovered in the piscivorous snail Conus catus using '"I-GVIA binding to rat brain membrane to guide fractionation of crude venom, and cloning to isolate the expressed gene products from venom duct tissue.

Results: Four new o-conopeptides (CVIA-D) were discovered in trace quantities in the crude venom. CVIA-D resemble most closely MVIIA, with loop 4 ofCVID showing most sequence divergence. The rank order of potency at the N-type calcium channel (CVID = MVIIA>CVIA>CVIC>CVIB>MVIIC) was reserved at the P/Q-type calcium channel. This series ofpeptides inhibited the electrically-induced contractile activity in rat vas deferens with potencies that correlated with displacement of '"I-GVIA binding, indicating that CVIA-D are functional inhibitors of N-type calcium channel activity. Of the peptides studies, CVID was the most selective for N-type versus P/Q-type calcium channels. The three-dimensional 'H NMR structure of CVID showed that loop 4 stabilised the structure of loop 2, a factor that may contribute to its ability to discriminate among neuronal calcium channels.

Conclusions: The co-conopeptides from C. catus represent an important new series of neuronal calcium channel inhibitors. The high N-type selectivity displayed by CVID (AM336) indicates this pep-tide may have potential in the treatment of pain.

Acknowledgments: Research supported by AMRAD Operations Pty Ltd and Auslndustry, under the Generic Technology component of the Industry Research and Development Act 1986, Australia.


Yasuyuki Honma. Michiaki Yamakage*, Akiyoshi Namiki, Dept. ofAnesthesiology Sapporo Medical Univ School of Medicine, Sapporo 060, Japan

Aim of Investigation: We hypothesized that abnormal activity and adrenergic sensitivity in injured dorsal root ganglion (DRG) neurons are due to an intrinsic alteration of the cell body membrane. We investigated the effects of adrenergic stimulus on the activities ofCa^ and K* channels of DRG neurons in a rat chronic constriction injury (CCI) model.

Methods: The protocol of this study was approved by our institute. CCI was made by loose ligations of the left sciatic nerve in male Wistar rats. After the ligation, withdrawal latencies from thermal stimuli in the hind paw were assessed to confirm hyperalgesia, and then the DRGs at L4-5 levels were separated. The DRG neurons were dissociated with collagenase and incubated for 3-10 hr at 37C. Inward Ca^ currents (IBa, Ba^ as a charge carrier) and outward K.^ currents were measured by whole-cell patch clamp recording at room temperature. After control measurement of both currents, norepinephrine (NE) was administered and the change of both peak currents were measured.

Results: a 2-adrenergic stimulus by 10 u.m NE inhibited IBa of DRGs in the CCI model by 42%, whereas it enhanced the IBa by 18% in control animals. The inhibitory effect ofNE disappeared by pretreatment with the N-type VDCC antagonist &>-conotoxin GVIA. o'2-adrenergic stimulus also inhibited outward K4 currents by 24% in the CCI model, while it had no effect on the currents in control animals. The inhibitory effect ofNE was blocked by pretreatment with the Ca^-activated K* (Kca) channel antagonist cha-rybdotoxin.

Conclusions: The NE-induced inhibitory effects both on N-type VDCC and on K<:a channels in injured DRG neurons of the CCI model could lead to cell membrane depolarization, resulting in a spontaneous discharge of action potential and an increase in sensitivity to adrenergic stimulus.


Shigeki Yamaguchi*', Yasuhisa Okuda', Toshimitsu Kitajima', Yuichi Hori*, ' Dept of Anesthesiology,2 Dept of Physiology, Dokkyo Univ School of Medicine, Tochigi, 321-0293, Japan

Aim of Investigation: Miniature excitatory post-synaptic currents recorded from neurons in the superficial dorsal hom of rat spinal cord slices were presynaptically inhibited by bath application of an opioid receptor agonist [Met5] enkephalin'*. However, the precise mechanisms for presynaptic inhibitory action of opioid is not clear.

Methods: Neonatal rat spinal cord slices were prepared and stained with calcium indicator fura 2-AM. Presynaptic Ca2+ transient evoked by electrical stimulation ofpresynaptic fibers was recorded in the presence ofCNQX, APV, bicuculline and strychnine.

Results: The amplitude of Ca2+ transient was markedly inhibited by m-conotoxin GVIA and inhibited to a lesser extent by co-agatoxin VIA. Micromolar concentrations of [Met5] enkephalin reduced the magnitude ofpresynaptic Ca2+ transient. The inhibitory effect of [Met5] enkephalin on Ca2+ transient was slightly feeble in the presence of co-agatoxin VIA and feeble to a greater extent in (o-conotoxin GVIA.

Conclusion: These observations suggest that presynaptic action of [Met5] enkephalin is mediated via modulation of both P/Q- and N-type calcium channels. 1) Hon Y, et al. J Physiology 450 : 673-685


Athena Quo*. Jianling Wang*, Robert Elde* (SPON: George Wil-cox) Dept of Cell Biology and Neuroanatomy, Univ of Minnesota, Minneapolis, MN 55455, USA

Aim of Investigation: To investigate the effect ofneurotrophic factors on VR1 expression and its association with neuropeptides, 1B4 binding sites, and P2X} purinoceptor in primary afferent neurons during development.

Methods: Antisera against both C- and N- terminal peptides of VR1 were generated. RT-PCR was used to isolate full length of VR1 cDNA from dorsal root ganglion (DRG). Immunostaining and western blotting analysis were used to determine the expression of VR1 in both transiently transfected cells and native tissues. Sub-cellular distribution of the VR1 protein was determined by electron microscopy.

Results: VR1 synthesized in small and medium sized DRG neurons was transported to both central and peripheral processes of primary afferents. In superficial dorsal hom, there was much higher density of VR1-positive nerve fibers and terminals in the inner layer than in the outer portion of lamina II. This pattern of staining was regulated by neurotrophins during development. In both central and peripheral processes of primary afferent neurons, while the differential relationship of VR1 with substance P and calcitonin gene related peptide remained the same during development, the relationship of VR1 with trophic factors, 1B4 binding sites, and P2X3 purinoceptor changed substantially.

Conclusions: Our results suggest that 1) DRG neurons containing both VR1 protein and IB4-binding sites may potentially act as thermal nociceptors; 2) multiple forms of VR1 may exist; 3) VR1 expression may be regulated by neurotrophic factors; 4) VR1 may be involved in the development of inflammation Acknowledgments: Supported by grants from NIDA.


Andreas Leffler*. Klaus Schmidt, Marc Schneider*, Friedrich Metzger* and Martin Koltzenburg, Dept. of Neurology, D-97080 Wiirzburg, Germany.

Aim of Investigation: Nociceptors can be subdivided into two broad groups ofpeptidergic and non-peptidergic cells. Here we asked whether the subpopulation of non-peptidergic nociceptors that bind the lectin 1B4 respond to capsaicin.

Methods: Neurons from adult mice dorsal root ganglia were dissociated and cultured for 24 hours in serum free medium. Neurons were labeled with fluorescent 1B4 and studied with standard whole cell patch clamp techniques or measurement ofintracellular calcium transients using ratiometric fura-2 imaging. Results: 1B4 labeled small diameter cells (15-25 um). In patch clamp studies (43 cells) capsaicin (luM) evoked inward currents in 48% ofIB4-positive (n=23) and in 45% ofIB4-negative (n=20) cells. Current amplitudes in individual neurons ranged from 350-5600 pA in both populations. Calcium imaging (75 cells) elicited strong calcium transients in 25% of the IB4-positive (n=32) and 23% of the IB4-negative (n=43) cells. There was no difference in the mean increase of the mtracellular calcium concentration between both populations of neurons.

Conclusions: Patch clamp and calcium imaging studies concur that equal proportions of 1B4- positive and IB4-negative neurons in mice are excited by capsaicin. This contrasts with our finding that capsaicin-induced cobalt uptake in mice occurs only in the IB4-negative population of nociceptors.

Acknowledgments: Supported by DFG, SFB 353.


Klaus H Schmidt, Martin Koltzenburg, Dept. of Neurology, D-97080 Wiirzburg, Germany

Aim of Investigation: To assess in adult mice if the two non-overlapping subpopulations ofnociceptive neurons that either contain CGRP and respond to NGF or bind the lectin 1B4 and express receptors for GDNF, differ in their response to capsaicin.

Methods: Sensory neurons from dissociated dorsal root ganglia of adult mice were grown in culture in the absence of exogenous growth factors or with NGF (50 ng/ml) or GDNF (50 ng/ml) added for 1 or 6 days. Capsaicin (1 u.m) sensitivity was assessed by uptake of cobalt ions and neurons were also stained for CGRP and 1B4.

Results: After 24 hours in culture 29% of the neurons (n=484) were positive for 1B4, 30% for CGRP and 34% for capsaicin. Less than 1% of the capsaicin sensitive cells (n=165) were positive for 1B4, 68% were CGRP-positive and the reminder was negative for both markers. After 6 days in culture (n= 614) the percentage of CGRP-positive neurons dropped to 18% and cobalt uptake to 8%. NGF (538 cells) prevented this drop in CGRP-(30%) and capsaicin-sensitive neurons (39%) whereas GDNF (626 cells) only prevented the drop in capsaicin sensitivity (34%), but not that of CGRP (19%). The percentage of 1B4 positive neurons was neither affected by NGF (34%) nor GDNF (32%).

Conclusion: in adult mice the majority ofCGR?-, but none of the IB4-positive neurons show capsaicin-induced cobalt uptake. Cap-saicin-sensitivity and CGRP synthesis are differentially regulated by NGF and GDNF.

Acknowledgments: Supported by DFG, SFB 353.


Istvan Nagy'' and Humphrey Rang. 'Dept of Anaesthetics, Imperial College School of Medicine, St Mary's Hospital, London, W2 1NY, UK; ^ovartis Inst for Medical Sciences, 5 Gower Place, London, WC 1E6BN, UK.

Aim of Investigation: Cell lines transfected with the vanilloid receptor, VR1, respond to noxious heat stimulation, as well as to capsaicin, suggesting that the same channel might account for the sensitivity ofnociceptive sensory neurons to both stimuli. Here we used single-channel recording to study the relationship between heat- and capsaicin-sensitivity in sensory neurons.

Methods: Single-channel recordings in cell-attached and inside-out configuration were made from cultured dorsal root ganglion neurons from adult rats. The holding potential was -60mV.

Results: Heat stimuli (up to 50C) and capsaicin (2 uM) evoked channel activity in 64 out of 342 patches and in 17 out of 232 patches, respectively. Patches containing a single well-resolved channel were analysed:

  Amplitude (pA) Mean open time (ms)
Heat (n= 3) 3.8 0.2 0.2 0.02
Capsaicin (n=2) 2.7 0.2 0.6 0.2


In a series of 145 inside-out patches tested consecutively with heat and capsaicin, the distribution of heat- and capsaicin-sensitive channels was'

  Heat-sensitive Heat-insensitive Total
Capsaicin-sensitive 6 5 11 (7.5%)
Capsaicin-insensitive 20 114 134(92.5%)
Total 26(17.9%) 119(82.1%) 145(100%)


Conclusions: Though at the whole-cell level, capsaicin-sensitivity co-segregates with heat-sensitivity (Nagy & Rang, 1999), this is not the case at the single-channel level, suggesting that distinct molecular entities (possibly different post-translational modifications of the VR1 gene product) mediate the two responses. The different channel characteristics of heat- and capsaicin-activated channels are consistent with this possibility. Nagy I and Rang H P (1999) Neuroscience, 88, 995-997.


M.A. Wilk-Blaszczak*. A.J. Szczepek*, J.H. Butterfield', (Spon:M.G. Carry). *UT Southwestern Medical Center, Dept of Anesthe-siology, Dallas, TX 75235. 'Mayo Clinic, Rochester, MN 55905.

Aim of Investigation: To characterize the vanilloid receptor on HMC-1 cell line with electrophysiological methods.

Methods: The standard whole-cell patch-clamp technique was used to measure the inward current produced by application of capsaicin. The cells were voltage clamped at a holding potential -60 mV (pCLAMP software, Axon Instruments). The capsacin effect was measured as the maximal inward current after application. The extracellular solution included (in mM): 140.0 NaCI, 5.0 KC1, 2.0 CaCI;, 1.0 MgCl;, 10.0 HEPES, 15.0 glucose, pH 7.2; mtracellular (in mM): 135.0 Kglutamate, 10.0 NaCI, 7.0 MgCl;, 10.0 HEPES, 3.0 ATP, 0.1 GTP, pH 7.2. Capsaicin was pipetted directly into the bath in aliquots of 200(^1.

Results: In HMC-1 cell line, studied under voltage clamp conditions, the extracellular application of l^iM capsaicin produced a rapid inward current of average amplitude of230.8 (n=9). This current was associated with an increase in input conductance. This current desensitized rapidly in the presence of extracellular calcium, returning to control levels within 30sec. The action of capsaicin was specifically inhibited with the prior application of 10X excess of capsazepine.

Conclusions: The action of capsaicin in inducing neurogenic inflammation involves the activation of mast cells. The characterization of this channel will clarify the direct role of mast cells in this process.

Acknowledgments: Supported by NIH grant GM47721.


Zoltan Olah'*. Tamas Szabo', Peter Blumberg', Michael J. lada-rola'', (SPON: R. Traub), 'Neuronal Gene Expression Unit, NIDCR, laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, NIH, Bethesda, MD 20892, USA

Aim of Investigation: The dynamic effects of a potent vanilloid, resiniferatoxin (RTX), were studied at the behavioral, molecular, and biochemical levels. We investigated mechanisms regulating the vanilloid receptor/Ca-ionophore, VR1.

Methods: Subcutaneous administration of RTX, produced a rapid short-lived hyperalgesic period (within Ih), followed by a long lasting (~2 weeks) hypoalgesia in rats.

Results: We demonstrated that recombinant VR1 is a binding site for RTX. RT-PCR demonstrated high levels of VR1 mRNA in the dorsal root ganglion (DRG), but not in spinal cord (SQ). DNA-array assays of 588 mRNAs show that systemic administration of RTX induces de novo transcription of several genes. To study the interaction between agonist stimulation and receptor regulation Ca-uptake (over a 25 min period), and ['HJRTX binding experiments were carried out in DRG-neuron cultures. Normally, capsaicin treatment elevates Ca-uptake in these cells, however, RTX pre-treatment eliminated the Ca-influx. We noted that the algesic pep-tide, bradykinin (BK), potently inhibits the RTX-induced down-regulation of the capsaicin stimulated Ca-influx. A membrane permeable form ofcGMP, but not cAMP, was also a potent antagonist of RTX-induced down-regulation. This indicates that cGMP can mimic the blocking effect of bradykinin on RTX- induced downregulation of VR1 Ca-influx in DRG neurons.

Conclusions: The results with BK suggest that a signal transduction mechanism, operated by cGMP, may be involved in a pathway for down-regulation of VR1. Furthermore, VR1 is a target for convergence of several receptor coupled second messenger systems which regulate the sensitivity of the channel. These data suggest molecular mechanisms which contribute to hyperalgesia.


M. Petersen, A. Klusch', C. Belmonte", R.F. Schmidt, F.-K. Pierau+ and M. Valdeolmillos''^, Inst of Physiology, Univ ofWiir-zburg, D-97070 Wiirzburg, Germany, ^Institute de Neurociencias, Universidad Miguel Hemandez, 03550 Alicante, Spain, and ^ax-Planck-Institut fur physiologische und klinische Forschung, D-61231 Bad Nauheim, Germany.

Aim of Investigation: An involvement of the Bl bradykinin receptor in nociception under pathological conditions has been postulated. In bradykinin binding studies we demonstrated an induction of the Bl receptor in rat dorsal root ganglion neurons following sciatic nerve injury. The aim of this study was to determine whether these Bl bradykinin receptors are functional and whether they are co-expressed with B2 bradykinin receptors and with the capsaicin receptor VR1.

Methods: Following three days tight ligation of the sciatic nerve, ipsilateral dorsal root ganglia L4/L5 were isolated. After dissociation, cells were plated on coverslips and kept under standard culture conditions for one to three days. The intracellular Ca2+-concentration was measured by microfluometry. Neurons were loaded with the Ca2+-sensitive fluorescent chelator fura-2/AM. Thereafter they were exposed to [des-Arg'j-bradykinin (B 1 ago-nist; 1 to 10 u.m), bradykinin (B2 agonist; 1 ^M), and capsaicin (VR1 agonist; 1 u.m).

Results: Of the 58 neurons tested, 27 (47%) responded to the B2 receptor agonist with an increase in intracellular Ca2+-concentration. Five (9%) of all neurons tested responded to the Bl receptor agonist. All neurons responsive to the Bl agonist also responded to the B2 agonist and to the VR1 agonist.

Conclusions: These results indicate that there is a small population of dorsal root ganglion neurons expressing functional Bl bradykinin receptors following mechanical nerve injury. As there is a co-response to bradykinin and capsaicin we hypothetize that the Bl receptor-expressing neurons are nociceptors.

Acknowledgments: Supported by DFG Pe 299/3-2, SAF/97-0195, and the Max-von-Frey Gesellschaft.


M. Kress. S. Gunther, Institut f. Physiologic u. Experimentelle Pathophysiologie, Universitatsstr. 17, D-91054 Eriangen, Germany

Aim of Investigation: Underlying the heat sensitivity of nociceptors are slowly activating and inactivating heat-induced membrane currents that have been identified in a cellular model. Here we for the first time demonstrate sensitization of such heat-activated membrane currents that can be triggered by rises in intracellular calcium concentration.

Methods: Simultaneous measurements of membrane currents and of(Ca2+) in dorsal root ganglion (DRG) neurons were performed using the patch-clamp technique and microfluonmetry with the calcium indicator dye fura-2 which was loaded into the neuron via the patch pipette. Heat stimuli (step temperature rise to 45 C for 2 s) were applied to single neurons at 1 min intervals using a feedback controlled device (Dittert et al. J. Neurosci. Meth. 1998). ATP (up to 100 uM), capsaicin (1 uM), acidic extracellular pH (pH 5.6) or the calcium ionophore ionomycin (10 uM) were used to elevate [Ca2"],.

Results: Rises in [Ca "], induced a sensitization of the heat-activated current This sensitization recovered in parallel with the decline oftCa2"^ and there was a significant correlation of[Ca2+], and of the amplitude of heat-activated currents (r=0.59, p<0.05).

Conclusion: Our results suggest that substances that introduce rises in [Ca2+], yield a sensitization of heat-activated currents in noci-ceptive neurons. This hints to intracellular Ca2+ as a trigger for plastic changes not only in central neurons but also in the peripheral nociceptive system which may account for heat hyperalgesia during inflammation.

Acknowledgments: Supported by Sanderstiftung and DFG


J.A.M. Smith*. H.K. Raymon*, J. Zhou*, N. Verity*, D.W.Y. Sah*, R. Johnson* and K.R. Bley. Roche Bioscience. 3401 Hillview Ave., Palo Alto, CA 94304 and Signal Pharmaceuticals, 5555 Oberlin Drive, San Diego, CA 92121 USA

Aim of Investigation: Low pH buffer depolarizes and excites nociceptive sensory neurons through activation of a fast, rapidly inactivating, Na*-selective current followed by a non- or slowly-inacti-vating non-selective cation current. A concomitant low pH-evoked increase in cytosolic [Ca2+] ([Ca2*],) can be observed. In this study the properties of low pH- and capsaicin-evoked increases in [Ca2+], in rat dorsal root ganglion (DRG) neurons were compared with those in a novel immortalized human sensory neuronal cell line, HD10.6 (Raymon et al., J. Neurosci., submitted)

Methods: HD10.6 cells were neuronally differentiated for >12 days. Cultures of embryonic rat DRG neurons were prepared using a modification of Smith et al., 1998 (Br. J. Pharmacol. 124, 513). Cells were loaded with fluo-3AM prior to measurement of changes in [Ca2"],, in single cells, using a video camera-based imaging system, and in cell populations, using a fluorescence imaging plate reader (FLIPR). The stimulus was Mg^-free Hanks Balanced Salt solution with 10 mM MES (HBSS), at different pH.

Results: In HD10.6 and rat DRG neurons, HBSS buffer at pH 6.9 to 6.5 evoked a transient increase in [Ca2*],. pH < 6.5 evoked a biphasic increase in [Ca2*], with distinct transient and sustained components. The pH response characteristics were similar in single cells and cell populations. HD10.6 neurons also exhibited transient and sustained whole-cell current responses to low pH. The transient and sustained components, of both [Ca2*], and whole-cell current responses, were blocked by amiloride (100 u.m) and ruthenium red (10 u.m), respectively, but not capsazepine (10 u.m). In contrast, capsaicin (500 nM) evoked a robust capsazepine-sensitive sustained increase in [Ca2"], in both DRG and HD10.6 neurons.

Conclusions: The characteristics of low pH and capsaicin-evoked increases in [Ca2*], were similar between HD10.6 and embryonic rat DRG neurons. Thus HD10.6 cells may represent a valuable tool for studying some of the properties of human nociceptors.


Sinerik N. Ayrapetyan and Armine A. Danielvan. Biophysics Center of Armenian NAS, Hasratian str. 7, Yerevan, 375014, Armenia

Aim of Investigations: The metabolic pathway through which abnormal excitation could produce nociceptive signal in nerve endings was studied. Earlier it was shown that the protein molecules on the cell membrane with receptive, enzymatic and ionophoretic properties are in functionally active and inactive states. The ratio of active and inactive molecules in the membrane is determined by the sizes of active surface membrane area depending on cell hy-dration.

Methods: The influence of both the abnormal stimulation and NO on cell hydration, radioactive ouabain binding, Na-K pump and Na:Ca exchange, intracellular messengers in rats' nervous tissues were studied.

Results: It was shown that the abnormal excitation of neuronal cells leads to the abnormal hydration. The Na and Ca enrichment of the cell in the result of abnormal discharge leads to Ca-dependent inhibition of Na-K ATPase. The Ca-dependcnt activation of NO production leads to the cGMP dependent removal of intracellular Ca through the activation of Na:Ca exchange. Thus cell hydration is a messenger through which the metabotropic effect of nociceptive signals is realized. Conclusions: The main consideration is that cell hydration could determine membrane excitation. Therefore factors leading to the elevation of cell hydration could promote nociceptive signal generation. The dehydration has an opposite effect on it.


Elvira de la Pena*. Salvador Sala*, Robert F. Schmidt, Carlos Belmonte (SPON: R. Rodriguez), Institute de Neurociencias, Uni-versidad Miguel Hemandez, Campus de San Juan, Alicante, Spain.

Aim of Investigation: To study the effect ofelastoviscous solutions ofhylans (hyaluronan derivatives) used for intra- articular treatment of arthritic pain on the response characteristics of native stretch-activated ion channels from adult Xenopus laevis oocytes.

Methods: Patch-clamp recordings in the cell-attached configuration were performed from Xenopus oocytes in Barth's medium (control condition) and with hylans of different elastoviscosity (Biomatrix, Inc. USA). For mechanical stimulation monitored suction was applied through the microelectrode.

Results: When suction was applied, activity of stretch activated channels was recorded. Similar values of conductance (~30pS) and reversal potential (near OmV) were found in control and hylan solutions. The activity of the channels (N.po) was significantly r educed in the presence of hylan G-F 20 (Synvisc*), a clinically used hylan product containing 0.8% polymer and consisting of 80% hylan A (MW-6M) and 20% hylan B gel. A somewhat weaker effect was obtained with an elastoviscous solution of hylan A (MW-6M) with the same polymer concentration (0.8%) but without hylan B gel. In contrast, when a nonelastoviscous 0.8% solution of hylan A (MW-0.1 million) was used, no effect was observed. The activity was also reduced by 30 u.m streptomycin, a blocker ofmechosensitive channels.

Conclusion: Stretch-activated channels which are presumably involved in mechanotransduction have a decreased mechanical sensitivity in the presence of elastoviscous solutions ofhylans, but not in the presence of nonelastoviscous solutions of hylan of the same concentration. This may explain the analgesic effect ofin-tra-articular injection of elastoviscous solutions ofhylans in reducing knee joint mechano-nociceptors sensitivity in animals and arthritic patients.

Acknowledgments: Supported by a grant ofBiomatrix, Inc., Ridge-field, New Jersey, USA.


Junichi Yagi''. Naoki Hirai*', Rhyuji Sumino, 'Dept. of Physiology, Kyorin Univ. School of Medicine, Tokyo, 181-8611, ^Dept. of Physiology, Nihon Univ. School of Dentistry, Chiyoda-ku, Tokyo 101-0062, JAPAN

Aim of Investigation: It has been shown that ZD7288 (Zeneca Pharmaceuticals), a bradycardic agent, inhibits a hyperpolarization-activated current (/h) in cardiac cells and certain neurons. 4, which is a slowly activating inward cation (Na* and K*) current evoked by hyperpolarization, is also called the 'pacemaker current' because a depolarizing influence caused by activation of 4 during an afterhyperpolarization accelerates neuronal firing discharges. In this study we investigated the effects of ZD7288 on 4 and neuronal excitability in rat dorsal root ganglion (DRG) neurons.

Methods: Bilateral L4 and [-5 DRGs with sciatic nerves attached were excised from adult (6- to 12-wk old) rats anesthetized deeply with pentobarbital sodium (80mg/kg, i.p.). Whole cell patch-clamp recording of DRG neurons with their axons was carried out.

Results: While the amplitude of 4 was large in Aa/p-type DRG neurons, it was small in A6- and C-type neurons. ZD7288 inhibited /h selectively. In Aci/p-type DRG neurons, ZD7288 induced a small hyperpolarization of the membrane and reduced the number of frequency of repetitive action potentials evoked by a depolarizing current pulse.

Conclusions: It is possible that ZD7288-induced inhibition of 4 results in reduction of firing discharges mainly in Aa/p-type DRG neurons, which seem to possess a high density of 4 channels. These findings suggest that ZD7288 might reduce hyperalgesia by decreasing the discharge frequency of abnormally active DRG neurons after nerve injury.

Acknowledgments: Supported by Research Projection of Kyorin Univ and the Sato Foundation of Nihon Univ.

9th WORLD CONGRESS ON PAIN, 1999, Vienna, Austria, p.131 - 138


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