GABA(B) receptor-mediated selective peripheral analgesia by the non-proteinogenic amino acid, isovaline.

Peripherally restricted analgesics are desirable to avoid central nervous system (CNS) side effects of opioids. Nonsteroidal anti-inflammatory drugs produce peripheral analgesia but have significant toxicity. GABA(B) receptors represent peripheral targets for analgesia but selective GABA(B) agonists like baclofen cross the blood-brain barrier. Recently, we found that the CNS-impermeant amino acid, isovaline, produces analgesia without apparent CNS effects. On observing that isovaline has GABA(B) activity in brain slices, we examined the hypothesis that isovaline produces peripheral analgesia mediated by GABA(B) receptors. We compared the peripheral analgesic and CNS effect profiles of isovaline, baclofen, and GABA (a CNS-impermeant, unselective GABA(B) agonist). All three amino acids attenuated allodynia induced by prostaglandin E2 injection into the mouse hindpaw and tested with von Frey filaments. The antiallodynic actions of isovaline, baclofen, and GABA were blocked by the GABA(B) antagonist, CGP52432, and potentiated by the GABA(B) modulator, CGP7930. We measured Behavioural Hyperactivity Scores and temperature change as indicators of GABAergic action in the CNS. ED(95) doses of isovaline and GABA produced no CNS effects while baclofen produced substantial sedation and hypothermia. In a mouse model of osteoarthritis, isovaline restored performance during forced exercise to baseline values. Immunohistochemical staining of cutaneous layers of the analgesic test site demonstrated co-localization of GABA(B1) and GABA(B2) receptor subunits on fine nerve endings and keratinocytes. Isovaline represents a new class of peripherally restricted analgesics without CNS effects, mediated by cutaneous GABA(B) receptors.

Modulation of flash-induced photosystem II fluorescence by events occurring at the water oxidizing complex.

The mechanism of flash-induced changes with a periodicity of four in photosystem II (PSII) fluorescence was investigated with the aim of further using fluorescence measurements as an approach to studying the structural and functional organization of the water-oxidizing complex (WOC). The decay of the flash-induced high fluorescence state of PSII was measured with pulse amplitude modulated fluorometry in thylakoids and PSII enriched membrane fragments. Calculated QA- decay was well described by three exponential decay components, reflecting QA- reoxidation with halftimes of 450 and 860 micros, 2 and 7.6 ms, and 111 and 135 ms in thylakoids and PSII membranes, respectively. The effect of modification of the PSII donor side by changing pH or by removal of the extrinsic 17 and 24 kDa proteins on period four oscillations in both maximum fluorescence yield and the relative contribution of QA- reoxidation reactions was compared to flash-induced oxygen yield. The four-step oxidation of the manganese cluster of the WOC was found to be necessary but not sufficient to produce modulation of PSII fluorescence. The capacity of the WOC to generate molecular oxygen was also required to observe a period four in the fluorescence; however, direct quenching by oxygen was not responsible for the modulation. Potential mechanisms responsible for the periodicity of four in both maximum fluorescence yield pattern and flash-dependent changes in proportion of centers with different QA- reoxidation rates are discussed with respect to intrinsic deprotonation events occurring at the WOC.

Induction of luminol chemiluminescence by the manganese cluster of the photosystem II water-oxidizing complex to the S0, S1, S2, and S3 states.

Luminol chemiluminescence (CL) (lambda(max) = 425 nm) induced by the manganese cluster of photosystem II (PSII) (t(max) = 1-5 min) along with CL induced by mono-, di-, or tetranuclear manganese coordination complexes (t(max) = 2-40 s) is observed when either 200 mM sodium phosphate or 1 M Tris-HCl + 200 microM EDTA are present in the reaction medium (pH 8.5) containing peroxidase. This light emission is not observed when Tris is used in the reaction medium without EDTA. The yield of a given CL without peroxidase is 5%-20% of that with peroxidase. The peroxidase-dependent CL is inhibited by catalase (I50 = 0.14-0.17 mu M), while the peroxidase-independent CL is not inhibited by 100 mM ethanol, 1 mM NaN3, 20 mu M Cyt c, or 0.6 mu M catalase. The CL induced by the Mn cluster of the water-oxidizing complex (WOC) in the S3 or S2 state exceeds that of lower S states by 15-20-fold. The magnitude of CL induced by Mn complexes is dependent on the ligand type of the complex. The ligand types of the Mn complexes and the WOC in different S states are ranked according to the magnitude of the induced CL: 1,10-phenanthroline > 2.2'-bipyridine > WOC (S3 or S2) > hydrotris(pirazolyl) borate > WOC (S0-2 or S0, S1) > 1,4,7-triazacyclononane. It is concluded that CL is caused by H2O2 formed as a result of the oxidation of luminol by triplet molecular oxygen. The Mn cluster of WOC and manganese coordination complexes, acting as catalysts of this reaction, show oxidase activity. Upon S2-S3 or S1-S2 transition, changes occur in the ligand environment of the Mn cluster of WOC influencing the induction of luminol CL.