Purinergic-Glycinergic Interaction in Neurodegenerative and Neuroinflammatory Disorders of the Retina.

Neurodegenerative-neuroinflammatory disorders of the retina seriously hamper human vision. In searching for key factors that contribute to the development of these pathologies, we considered potential interactions among purinergic neuromodulation, glycinergic neurotransmission, and microglia activity in the retina. Energy deprivation at cellular levels is mainly due to impaired blood circulation leading to increased release of ATP and adenosine as well as glutamate and glycine. Interactions between these modulators and neurotransmitters are manifold. First, P2Y purinoceptor agonists facilitate reuptake of glycine by glycine transporter 1, while its inhibitors reduce reverse-mode operation; these events may lower extracellular glycine levels. The consequential changes in extracellular glycine concentration can lead to parallel changes in the activity of NR1/NR2B type NMDA receptors of which glycine is a mandatory agonist, and thereby may reduce neurodegenerative events in the retina. Second, P2Y purinoceptor agonists and glycine transporter 1 inhibitors may indirectly inhibit microglia activity by decreasing neuronal or glial glycine release in energy-compromised retina. These inhibitions may have a role in microglia activation, which is present during development and progression of neurodegenerative disorders such as glaucomatous and diabetic retinopathies and age-related macular degeneration or loss of retinal neurons caused by thromboembolic events. We have hypothesized that glycine transporter 1 inhibitors and P2Y purinoceptor agonists may have therapeutic importance in neurodegenerative-neuroinflammatory disorders of the retina by decreasing NR1/NR2B NMDA receptor activity and production and release of a series of proinflammatory cytokines from microglial cells.

Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides.

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.

Gelsemine, a principal alkaloid from Gelsemium sempervirens Ait., exhibits potent and specific antinociception in chronic pain by acting at spinal α3 glycine receptors.

The present study examined the antinociceptive effects of gelsemine, the principal alkaloid in Gelsemium sempervirens Ait. A single intrathecal injection of gelsemine produced potent and specific antinociception in formalin-induced tonic pain, bone cancer-induced mechanical allodynia, and spinal nerve ligation-induced painful neuropathy. The antinociception was dose-dependent, with maximal inhibition of 50% to 60% and ED50 values of 0.5 to 0.6 µg. Multiple daily intrathecal injections of gelsemine for 7 days induced no tolerance to antinociception in the rat model of bone cancer pain. Spinal gelsemine was not effective in altering contralateral paw withdrawal thresholds, and had only a slight inhibitory effect on formalin-induced acute nociception. The specific antinociception of gelsemine in chronic pain was blocked dose-dependently by the glycine receptor (GlyR) antagonist strychnine with an apparent ID50 value of 3.8 µg. Gelsemine concentration-dependently displaced H(3)-strychnine binding to the membrane fraction of rat spinal cord homogenates, with a 100% displacement and a Ki of 21.9µM. Gene ablation of the GlyR α3 subunit (α3 GlyR) but not α1 GlyR, by a 7-day intrathecal injection of small interfering RNA (siRNA) targeting α3 GlyR or α1 GlyR, nearly completely prevented gelsemine-induced antinociception in neuropathic pain. Our results demonstrate that gelsemine produces potent and specific antinociception in chronic pain states without induction of apparent tolerance. The results also suggest that gelsemine produces antinociception by activation of spinal α3 glycine receptors, and support the notion that spinal α3 glycine receptors are a potential therapeutic target molecule for the management of chronic pain.

Pathway-dependent modulation by P2-purinoceptors in the mouse retina.

Adenosine trisphosphate (ATP) activates purinoceptors and acts as a neurotransmitter in the nervous system. In the retina, we previously reported that the immunohistochemical distribution of the subset of P2-purinoceptors differs between the ON and OFF pathways. Here, we investigated whether ATP activates P2-purinoceptors and modulates the physiological function of the mouse retina. We also examined if signal processing by P2-purinoceptors is pathway specific. Results showed that ATP activated both ON- and OFF-cholinergic amacrine cells. However, responses in OFF-cholinergic amacrine cells were greater than those in ON-cholinergic amacrine cells. Pharmacological studies in OFF-cholinergic amacrine cells showed that the response of OFF-cholinergic amacrine cells is mediated P2X(2)-purinoceptors. Further, ATP increased gamma-aminobutyric acid (GABA)ergic inhibitory postsynaptic currents (IPSCs) in OFF- but not ON-cholinergic amacrine cells. The increase in GABAergic IPSCs was mediated by P2-purinoceptors. P2-purinoceptor-mediated signals suppressed OFF ganglion cells but activated ON ganglion cells. Our findings indicate that ATP physiologically modulates signal processing of the ON and OFF pathways in a pathway-specific manner through P2-purinoceptors.

Identification of UDP-glucuronosyltransferases involved in the human hepatic metabolism of GV150526, a novel glycine antagonist.

The major metabolic pathway for elimination of GV150526 is by glucuronidation exerted by glucuronosyl transferases (UGTs). Potential exists for the modification of GV150526 pharmacokinetics by drugs capable of inhibiting the glucuronidation of GV150526. Using human liver microsomes, 44 compounds were screened for inhibition of GV150526 glucuronidation. These compounds were selected because they are extensively glucuronidated themselves or are used as concomitant medication in the treatment of acute stroke. For 11 compounds out of the 44, full inhibition kinetics were performed to determine their Ki-value and mechanism of inhibition. To predict possible in vivo drug-drug interactions, the theoretical percentage of inhibition (i) was determined, based on in vitro determined Ki-values, and the expected Cmax plasma levels of GV150526 and the inhibitor. Of the 11 compounds examined, only propofol had an i-value of 6.6; for all other compounds i-values were lower than 2.1. These results indicate that although in vitro inhibition is observed, the likelihood of in vivo drug-drug metabolic interactions occurring is low. The inhibition results suggest that in addition to UGT1A1, also UGT1A3, UGT1A8/9, and UGT2B4 are involved in the glucuronidation of GV150526. The involvement of UGT1A1 and UGT1A8/9 was confirmed from studies using cDNA expressed human UGT cell lines.

Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform.

Using the whole-cell patch-clamp technique, we demonstrate glycine-induced currents in oligosphere-derived oligodendrocyte progenitors cultured from newborn rats. Similar inward currents are also triggered by beta-alanine and taurine, two established glycine receptor agonists. In our recording conditions, glycine-gated currents in oligodendrocyte progenitors reverse about 0 mV and are reversibly inhibited by the glycine competitive antagonist strychnine, the Cl- channel blocker picrotoxinin and the non-competitive antagonist cyanotriphenylborate. The oligodendrocyte progenitors glycine receptor (GlyR) differs from the corresponding neuronal receptor: [3H]strychnine binding data and the strychnine inhibition curve of glycine-induced currents in oligodendrocyte progenitor cultures suggest the existence of two strychnine binding sites on the oligodendroglial GlyR. Using total RNA isolated from oligodendrocyte progenitors cultures, reverse transcription-polymerase chain reaction analysis of glycine receptor subunit expression shows the presence of alpha2 and beta subunits and immunocytochemical stainings confirm that this GlyR contains an alpha subunit which is not alpha1. The molecular structure of the oligodendroglial GlyR could be either homopentameric alpha2 or heteromeric alpha2beta but in both cases, the sequence of the alpha2 or beta subunits have to be different from the known neuronal sequences in order to explain, respectively, the cyanotriphenylborate (alpha2) and picrotoxinin (beta) sensitivities. This work thus demonstrates that GlyR are expressed by oligodendrocytes obtained not only from spinal cord but also from supraspinal structures. The pharmacological properties and presumably the molecular structure of oligodendroglial GlyR are original. The physiological meaning of the presence of such receptors on developing and mature oligodendrocytes remains unknown.