resiniferatoxin and chelerythrine

We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH.. Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals.. Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of TRPV1 receptors via a PKC-dependent pathway, which leads to the development of TH mimicking what occurs in chronic ischemic patients with severe acidosis. More importantly, peripheral P2Y1 receptors play a pivotal role in this process, suggesting a novel peripheral mechanism underlying the development of TH in these patients.

Topics: Acids; Acrylamides; Adenosine Diphosphate; Animals; Benzophenanthridines; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Diterpenes; Hindlimb; Hot Temperature; Hyperalgesia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Injections; Ion Channels; Ischemia; Pain; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2Y1; Sodium Chloride; Thrombosis; Tissue Extracts; TRPV Cation Channels

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Topics: Alkaloids; Aniline Compounds; Arachidonic Acids; Benzophenanthridines; Calcium; Capsaicin; Cell Line; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Fluorescence; Fluorometry; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Phenanthridines; Polycyclic Sesquiterpenes; Polyunsaturated Alkamides; Protein Kinase C; Receptors, Drug; Ruthenium Red; Sesquiterpenes; Time Factors; Xanthenes

The extension of cellular processes from the oligodendrocyte soma is an early and critical event in myelin formation. Previous reports from this laboratory have implicated a role for protein kinase C (PKC) as an important intracellular mediator of this critical step in myelinogenesis. In the current study, the regrowth of fibers by adult human oligodendrocytes was examined and was found to be significantly enhanced by the PKC stimulator, 4 beta-phorbol-12,13-didecanoate (PDB); this was accompanied by a 400-500% increase in oligodendroglial PKC activity. In contrast to other cell types, the increased PKC activity in oligodendrocytes was not followed by subsequent down-regulation of the enzyme. The role of PKC in oligodendroglial process formation was further demonstrated by the ability of inhibitors of PKC to block the basal- or PDB-enhanced fiber outgrowth. As well, studies employing isoform-specific agonists implicated PKC alpha as the major determinant of fiber outgrowth by oligodendrocytes. The potential significance of PKC in myelin formation was further underscored by the observation that the synthesis of myelin basic protein, a prerequisite component for myelinogenesis, was increased by 2-fold in PDB-treated oligodendrocytes. Collectively, these observations suggest that PKC, in particular the alpha isoform, constitutes an important mediator in the initiation of myelin formation.

Topics: Adult; Alkaloids; Animals; Base Sequence; Benzophenanthridines; Brain; Cells, Cultured; Diterpenes; Female; Humans; Isoenzymes; Molecular Sequence Data; Myelin Sheath; Nerve Tissue Proteins; Oligodendroglia; Phenanthridines; Phorbol Esters; Protein Kinase C; Rats; Rats, Wistar; Staurosporine