Zoltan Olah

Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control

Control of cancer, neuropathic, and postoperative pain is frequently inadequate or compromised by debilitating side effects. Inhibition or removal of certain nociceptive neurons, while retaining all other sensory modalities and motor function, would represent a new therapeutic approach to control severe pain. The enriched expression of transient receptor potential cation channel, subfamily V, member 1 (TRPV1; also known as the vanilloid receptor, VR1) in nociceptive neurons of the dorsal root and trigeminal ganglia allowed us to test this concept. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya induces calcium cytotoxicity by opening the TRPV1 ion channel and selectively ablates nociceptive neurons. This treatment blocks experimental inflammatory hyperalgesia and neurogenic inflammation in rats and naturally occurring cancer and debilitating arthritic pain in dogs. Sensations of touch, proprioception, and high-threshold mechanosensitive nociception, as well as locomotor function, remained intact in both species. In separate experiments directed at postoperative pain control, subcutaneous administration of RTX transiently disrupted nociceptive nerve endings, yielding reversible analgesia. In human dorsal root ganglion cultures, RTX induced a prolonged increase in intracellular calcium in vanilloid-sensitive neurons, while leaving other, adjacent neurons unaffected. The results suggest that nociceptive neuronal or nerve terminal deletion will be effective and broadly applicable as strategies for pain management.

Physiologic and antinociceptive effects of intrathecal resiniferatoxin in a canine bone cancer model

Background:Resiniferatoxin is a potent capsaicin analog. Intrathecal administration leads to selective, prolonged opening of the transient receptor potential V1 ion channel, which is localized mainly to C-fiber primary afferent nociceptive sensory neurons. Following work in laboratory animals, the authors explored the use of intrathecal resiniferatoxin to control spontaneous bone cancer pain in companion (pet) dogs. Methods:Normal canine population: Behavioral testing was performed to establish baseline paw withdrawal latency; subsequently, general anesthesia was induced and resiniferatoxin was administered intrathecally while hemodynamic parameters were recorded. Behavior testing was repeated for 12 days after administration of resiniferatoxin. Clinical canine population: Twenty companion dogs with bone cancer pain were recruited. The animal’s baseline level of discomfort and analgesic use were recorded. Resiniferatoxin was administered intrathecally and hemodynamic parameters were monitored while the dogs were under general anesthesia. Dogs were reevaluated up to 14 weeks after resiniferatoxin administration. Results:Normal canine population: In the first minutes after resiniferatoxin injection, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate from baseline. Two days after injection, limb withdrawal latencies increased to the point of cutoff in the dogs that received at least 1.2 &mgr;g/kg resiniferatoxin. Clinical canine population: From baseline, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate after resiniferatoxin injection. Comfort scores were significantly improved at 2, 6, 10, and 14 weeks after resiniferatoxin administration (P < 0.0001). There was decreased or discontinued use of supplemental analgesics in 67% of the dogs 2 weeks after resiniferatoxin administration. Conclusions:Intrathecal resiniferatoxin elicits transient hemodynamic effects. In controls, a profound and sustained blockade of thermal stimuli is produced in a dose-dependent fashion. Similar administration in dogs with bone cancer produces a prolonged antinociceptive response.

Protein Kinase Cα Is Required for Vanilloid Receptor 1 Activation EVIDENCE FOR MULTIPLE SIGNALING PATHWAYS

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca2+ uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKCα in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCβ, -δ, -ε, and −ζ. Loss of PKCα correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKCα, but not PKCε. These studies suggest that PKCα in sensory neurons may elicit or enhance pain during inflammation or ischemia.

Protein Kinase C ϵ Subcellular Localization Domains and Proteolytic Degradation Sites A MODEL FOR PROTEIN KINASE C CONFORMATIONAL CHANGES

Protein kinase C (PKC) ε has been found to have unique properties among the PKC isozymes in terms of its membrane association, oncogenic potential, and substrate specificity. Recently we have demonstrated that PKCε localizes to the Golgi network via its zinc finger domain and that both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKCε, a series of NIH 3T3 cell lines were created, each overexpressing a different truncated version of PKCε. The overexpressed proteins each were designed to contain an ε-epitope tag peptide at the COOH terminus to allow ready detection with an antibody specific for the tag. The subcellular localization of the recombinant proteins was analyzed by in vivo phorbol ester binding, immunocytochemistry, and cell fractionation followed by immunoblotting. Results revealed several regions of PKCε that contain putative subcellular localization signals. The presence either of the hinge region or of a 33-amino-acid region including the pseudosubstrate sequence in the recombinant proteins resulted in association with the plasma membrane and cytoskeletal components. The catalytic domain was found predominantly in the cytosolic fraction. The accessibility and thus the dominance of these localization signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation sites also were identified. The susceptibility of the overexpressed proteins to proteolytic degradation was dependent on the protein conformation. Based on these observations, a model depicting the interaction and hierarchy of the suspected localization signals and proteolytic degradation sites is presented.

Cell-penetrating peptide exploited syndecans.

Cell-penetrating peptides (CPPs) are short peptides capable of translocating across the plasma membrane of live cells and transporting conjugated compounds intracellularly. Fifteen years after discovering the first model cationic CPPs, penetratin and TAT, CPP internalization is still challenging many questions. Particularly it has been unknown whether CPPs enter the cells with or without mediation of a specific surface receptor. Here we report that syndecan-4, the universally expressed isoform of the syndecan family of transmembrane proteoglycans, binds and mediates transport of the three most frequently utilized cationic CPPs (penetratin, octaarginine and TAT) into the cells. Quantitative uptake studies and mutational analyses demonstrate that attachment of the cationic CPPs is mediated by specific interactions between the heparan sulfate chains of syndecan-4 and the CPPs. Protein kinase C alpha is also heavily involved in the uptake mechanism. The collected data give the first direct evidence on the receptor-mediated uptake of cationic CPPs and may replace the long-thought, but already contradicted membrane penetration hypothesis. Thus our study might give an answer for a decade long debate and foster the development of rationalized, syndecan-4 targeted novel delivery technologies.

Peripherally induced resiniferatoxin analgesia.

&NA; Selective blockade of nociceptive pathways represents a mechanism‐based approach that has attracted a large variety of pharmacological and molecular investigations. A potential site for selective intervention is the primary afferent nociceptive nerve terminal. Binding of resiniferatoxin (RTX) to the vanilloid‐1 receptor (VR1) stimulates and then inactivates heat and vanilloid‐responsive nerve endings involved in heat and inflammatory pain signaling which can progress to localized degeneration of the peripheral ending followed by regeneration. Application of RTX directly to peripheral nerve endings produces a long term, reversible attenuation of nociceptive transmission. Heat hyperalgesia and mechanical allodynia were assessed prior to injection of RTX into the hindpaw (baseline) and at acute (minutes–hours) and more chronic (days–weeks) times after injection. Acutely, an inverse dose‐to‐pain response (guarding, licking) for RTX (0.0625–2.0 &mgr;g) occurs, followed by selective attenuation of peripheral pain transmission. Thermal nociception was decreased in a concentration‐dependent fashion and lasted up to 21 days, without impairing motor function. Administration of RTX blocked both inflammation‐induced hyperalgesia and spinal c‐Fos induction. The results demonstrate the efficacy and therapeutic potential of reversible, peripheral C‐fiber ‘inactivation’ for intermediate duration pain control.

Epidural resiniferatoxin induced prolonged regional analgesia to pain

Adequate treatment of cancer pain remains a significant clinical problem. To reduce side effects of treatment, intrathecal and epidural routes of administration have been used where appropriate to reduce the total dose of agent administered while achieving regional control. Resiniferatoxin (RTX), an ultrapotent capsaicin analog, gives long-term desensitization of nociception via C-fiber sensory neurons. We evaluate here the analgesic effect on rats of epidurally administered RTX, using latency of response to a thermal stimulus in unrestrained animals. Results were compared with those for systemically administered RTX. Vehicle or graded doses of RTX were injected subcutaneously (s.c.) or through an indwelling lumbar (L4) epidural catheter as a single dose. Both routes of application of RTX produced profound thermal analgesia, reaching a plateau within 4-6 h and showing no restoration of pain sensitivity over 7 days. Vehicle was without effect. For the epidural route, the effect was selective as expected for the targeted spinal cord region, whereas the subcutaneous administration of RTX had a generalized analgesic effect. At doses yielding a tripling of back paw withdrawal latency, epidural treatment was 25-fold more effective than the subcutaneous route of application. Consistent with the regional selectivity of the lumbar epidural route, the front paws showed no more effect than by systemic RTX treatment. Binding experiments with [3H]RTX provided further evidence of the segmental desensitization induced by epidural RTX. We conclude that epidural administration of RTX at the lumbar spinal level produces profound, long-lasting, segmental analgesia to C-fiber mediated pain in the rat.

Inhibition by H-7 of the protein kinase C prevents formation of brain edema in Sprague-Dawley CFY rats

The effect of the protein kinase C enzyme inhibitor H-7 was examined on the brain edema formation evoked by bilateral occlusion of the common carotid arteries in Sprague-Dawley rats of CFY strain. Brain edema was assessed by measurement of water and electrolyte contents of the brain. The results showed that pretreatment with H-7 reduced the extent of brain edema formation in a dose-dependent manner. The fact that H-7 treatment prevented the accumulation of water and certain electrolytes in the brain indicates that the protein kinase C may be activated not only in the neuronal structures but also in the microvessels during ischemia, which can lead directly or via certain calcium-mediated mechanisms to the opening of tight junctions resulting in the development of brain edema.

Cerebral ischemia induces transient intracellular redistribution and intranuclear translocation of the raf proto-oncogene product in hippocampal pyramidal cells

SummaryIn this report we describe changes in the intracellular redistribution of raf serine/threonine protein kinase (product of the raf proto-oncogene family) in hippocampal neurons following cerebral ischemia in Mongolian gerbils. For immunohistochemical localization studies polyclonal antisera specific for each of the A, B, and Raf-1 isotypes of raf, as well as a pan-raf antisera, were employed. Of these, only sera recognizing B-raf, as well as the general v-raf (raised against the conserved C-terminal region) were positive, indicating that B-raf is the major isotype in this neuronal region. Three different ischemie models were used (repeated 3 times for two min and single 5 or 15 min occlusions, of the common carotid arteries) to demonstrate that ischemie insult causes redistribution of raf protein kinase into the cell nucleus of hippocampal neurons. Increased amounts of raf protein in the nuclei of pyramidal cells following ischemia was confirmed by Western blot analysis of isolated nuclear fractionations. Moreover, an elevation in the level of nuclear raf protein also was detected in the contralateral (i.e. non-occluded hemisphere) neurons of CA1 and CA3 subfields 4 days after the ischemie insult indicating a possible transsynaptic increase in the amount of raf protein along with redistribution. The intranuclear translocation of the immunoreactive material started from the perinucleolar rim and with time extended throughout the nucleus. Enhanced levels and altered redistribution of the raf polypeptide in the nuclei of pyramidal cells of the CA3 subfleld appears to be reversible and returns to the normal level 12 days following the ischemic insult. In addition to triggering the above changes in the intracellular redistribution of raf, ischemie insult also caused an increase in the level of B-raf protein in reactive astrocytes.

Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells.

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca2+ accumulation, but not on cell phenotype.