No abstract

1 The responses of wide dynamic range spinal dorsal horn neurones to noxious mechanical stimulation of the ankle or knee joint were tested before and after spinal administration of the non-selective cyclooxygenase (COX) inhibitors, indomethacin and meclofenamic acid. Neither of these drugs altered the responses of these neurones to noxious mechanical stimulation. 2 Wind-up of a spinal nociceptive re¯ex evoked by electrical stimulation of the sural nerve at C-®bre strength was dose-dependently inhibited by intravenous administration of indomethacin, a non-selective COX inhibitor, and SC58125, a selective COX-2 inhibitor. Intrathecal administration of indomethacin also reduced the wind-up of this nociceptive re¯ex. 3 Western blot analysis of proteins extracted from normal rat spinal cord revealed the presence of both cyclo-oxygenase (COX)-1 and COX-2 proteins. 4 Immunocytochemistry of sections of normal rat spinal cord with speci®c COX-1 antiserum revealed little speci®c COX-1-like immunoreactivity in the grey matter. With the same antiserum, intense COX-1-like immunoreactivity was observed in the cytoplasm, nuclear membrane and axonal processes of small to medium sized (51000 mm 2 ) dorsal root ganglion (DRG) cell bodies. 5 Immunocytochemistry of sections of normal rat spinal cord incubated with speci®c COX-2 antiserum showed intense COX-2-like immunoreactivity (COX-2-li) in the super®cial dorsal horn of the spinal cord (laminae I and II) and around the central canal (lamina X). COX-2-li was also observed in some neurones in deep dorsal horn and in individual motor neurones in ventral horn. COX-2-li was not observed in the cell bodies of DRG. 6 Superfusion of the lumbar spinal cord of normal rats with arti®cial CSF and subsequent radioimmunoassay revealed the presence of prostaglandin D 2 (PGD 2 )5PGE 2 , but not PGI 2 (determined by measurement of the stable metabolite, 6-keto-PGF 1a ) or PGF 2a . 7 These data suggest that eicosanoids synthesized by an active COX pathway in the spinal cord of normal animals may contribute to nociceptive processing, but only when the spinal cord neurones are rendered hyperexcitable following C-®bre stimulation. Selective inhibition of one or both of the COX isoforms in normal animals may represent a novel target for spinal analgesia.

P2X receptors for adenosine 5'-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1-7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-meATP) and 2-methyl-thioadenosine 5'-triphosphate (2-meSATP), and using the antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2',5' disulphonic acid (PPADS). ATP (10 microM) evoked inward currents in approximately 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, approximately 4% showed a sustained response and approximately 10% showed a combination of transient and sustained components. Rapid application of ATP, alpha,beta-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-beta,gamma-methyleneadenosine 5'-triphosphate (1-beta,gamma-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.

No abstract

Whether podocytes are able to endocytose proteins is uncertain. We studied protein endocytosis in conditionally immortalized mouse and human podocytes using FITC-albumin by direct quantitative assay and by fluorescence microscopy and electron microscopy in mouse podocytes. Furthermore, in vivo uptake was studied in human, rat, and mouse podocytes. Both mouse and human podocytes displayed specific one-site binding for FITC-albumin with K d of 0.91 or 0.44 mg/ml and Bmax of 3.15 or 0.81 g/mg cell protein, respectively. In addition, they showed avid endocytosis of FITC-albumin with Km of 9.48 or 4.5 mg/ml and Vmax of 474.3 or 97.4 g⅐mg cell protein Ϫ1 ⅐h Ϫ1 , respectively. Immunoglobulin and transferrin were inefficient competitors of this process, indicating some specificity for albumin. Accumulation of endocytosed albumin could be demonstrated in intracellular vesicles by fluorescence confocal microscopy and electron microscopy. Endocytosis was sensitive to pretreatment with simvastatin.