Management of radioactive waste gases from PET radiopharmaceutical synthesis using cost effective capture systems integrated with a cyclotron safety system.

The emphasis on the reduction of gaseous radioactive effluent associated with PET radiochemistry laboratories has increased. Various radioactive gas capture strategies have been employed historically including expensive automated compression systems. We have implemented a new cost-effective strategy employing gas capture bags with electronic feedback that are integrated with the cyclotron safety system. Our strategy is suitable for multiple automated 18F radiosynthesis modules and individual automated 11C radiosynthesis modules. We describe novel gas capture systems that minimize the risk of human error and are routinely used in our facility.

Functional modulation of the human voltage-gated sodium channel NaV1.8 by auxiliary ß subunits.

The voltage-gated sodium channel Nav1.8 mediates the tetrodotoxin-resistant (TTX-R) Na+ current in nociceptive primary sensory neurons, which has an important role in the transmission of painful stimuli. Here, we describe the functional modulation of the human Nav1.8 α-subunit in Xenopus oocytes by auxiliary ß subunits. We found that the ß3 subunit down-regulated the maximal Na+ current amplitude and decelerated recovery from inactivation of hNav1.8, whereas the ß1 and ß2 subunits had no such effects. The specific regulation of Nav1.8 by the ß3 subunit constitutes a potential novel regulatory mechanism of the TTX-R Na+ current in primary sensory neurons with potential implications in chronic pain states. In particular, neuropathic pain states are characterized by a down-regulation of Nav1.8 accompanied by increased expression of the ß3 subunit. Our results suggest that these two phenomena may be correlated, and that increased levels of the ß3 subunit may directly contribute to the down-regulation of Nav1.8. To determine which domain of the ß3 subunit is responsible for the specific regulation of hNav1.8, we created chimeras of the ß1 and ß3 subunits and co-expressed them with the hNav1.8 α-subunit in Xenopus oocytes. The intracellular domain of the ß3 subunit was shown to be responsible for the down-regulation of maximal Nav1.8 current amplitudes. In contrast, the extracellular domain mediated the effect of the ß3 subunit on hNav1.8 recovery kinetics.

Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins?

The synthetic alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

Binding characteristics of the novel highly selective delta agonist, [3H]IIe5,6deltorphin II.

Following the description of the [3H]deltorphin II, it has been reported that the modification of deltorphin II with the substitution of Val5,6 residues by the more hydrophobic IIe5,6 residues leads to an increased affinity and selectivity. The IIe5,6 deltorphin II (Tyr-D-Ala-Phe-Gly-IIe-IIe-HH2) was tritiated by catalytic dehalogenation and labelled rat brain membrane sites with a Kd value of 0.40 nM and a Bmax of 121 fmol/mg protein. Competition binding experiments with various unlabelled subtype specific opioid receptor ligands resulted in mu/delta and kappa/delta selectivity ratios of 2400 and 18,000 respectively. Due to its high delta receptor affinity, delta selectivity and very low non-specific binding (< 20%), [3H]IIe5,6 deltorphin II, is a very useful tool for the identification and characterisation of delta opioid receptors.

A pore-lining glutamic acid in the rat olfactory cyclic nucleotide-gated channel controls external spermine block.

Spermine is a potent, voltage-dependent blocker of the olfactory cyclic nucleotide-gated channel from both the intracellular and extracellular sides. However, its sites of action are unknown. This study investigated the external spermine binding site in the rat CNCalpha3 subunit. Neutralization of a glutamic acid residue (E342Q) in the P-loop region eliminated voltage-dependence of block by externally applied spermine. The charge-conservative E342D mutation had little effect on spermine block. Thus, E342 forms the binding site for externally applied spermine. However, spermine remained a potent voltage-independent blocker of the E342Q mutant channel, suggesting that the mutation either created a novel binding site outside the membrane electrical field or that it dramatically changed the properties of the existing pore site.

Synthesis and binding characteristics of tritiated TIPP[psi], a highly specific and stable delta opioid antagonist.

The pseudopeptide H-Tyr-Tic psi [CH2-NH]Phe-Phe-OH (TIPP[psi]) is a delta opioid antagonist with high delta receptor affinity and unprecedented delta selectivity. TIPP[psi] was radiolabelled by catalytic tritiation of its precursor [Tyr(3',5'-I2)1]TIPP[psi]. The resulting radioligand, [3H]TIPP[psi], had a specific activity of 1.77 TBq/mmol (47.9 Ci/mmol) and showed high stability against enzymatic degradation. [3H]TIPP[psi] binding to rat brain membranes was saturable and Scatchard analysis indicated a single binding site with a Kd of 0.98 nM and a Bmax of 105.4 fmol/mg. A study of [3H]TIPP[psi] binding displacement by various receptor-selective opioids showed the expected rank order of potency (delta >> mu > kappa). [3H]TIPP[psi] represents an excellent new radioligand for delta receptor labelling studies in vitro and in vivo.

Synthesis and binding characteristics of the highly specific, tritiated delta opioid antagonist [3H]TIPP.

The recently discovered highly specific delta opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP) was radiolabelled by catalytic tritiation of its precursor [Tyr (3', 5'-I2)1]TIPP. The tritiated ligand labelled rat brain membrane binding sites with a Kd value of 0.64 nM and a Bmax of 82 fmol/mg protein. Binding competition experiments with various unlabelled mu-, delta- and kappa-opioid receptor ligands resulted in mu/delta and kappa/delta selectivity ratios of 600 and 9100, respectively, and thus confirmed the previously established high delta specificity of TIPP. Because of its high delta receptor affinity, extraordinary delta selectivity and low non-specific binding, [3H]TIPP represents an excellent new radioligand for the study of delta opioid receptor interactions.

Sharp vision: a prerequisite for compensation to myopic defocus in the chick?

PURPOSE: Compensatory responses to focusing errors imposed by spectacle lenses in chicks, tree shrews and primates leave little doubt that active emmetropization can occur, and debate is now centered on whether this process is uni-directional or bi-directional in nature. To provide further insight into this emmetropization process, the studies reported in this paper addressed the question of whether access to sharp vision is necessary for compensation to myopic defocus in the chick. METHODS: Two different experimental paradigms were used to address the above question: (A) Myopic defocus was imposed, either with +15 or +40 d lenses alone or with +15 D lenses on eyes made myopic by 7 days of form deprivation; these treatments result in a shift in the plane of focus of the eye (far point) to 6.67, 2.5 cm and approximately 3.5 cm resp., with only objects at or closer than these planes being in focus. The addition to the lenses of stand-off cones, either 2.5 or 5 cm in length, further limited access to (or precluded) sharp vision by controlling how closely the chicks could approach objects. One group that had sharp vision precluded also underwent optic nerve section. (B) A range of positive lenses (+15 to +65 D) were used on their own to impose myopic defocus; for the high power lenses, access to sharp vision was very restricted because of the close proximity of the new far point (1.54 cm for +65 D lens). Refractive errors and axial ocular dimensions were measured in all experiments. RESULTS: In the first study (A), preclusion of sharp vision not only prevented compensation but resulted in increased eye growth and myopia. This myopia, like form-deprivation myopia, was unaltered by optic nerve section surgery. Limiting but not precluding sharp vision resulted in partial compensation. In the second study (B), good compensation was observed with the +15 D lens but compensation progressively declined for higher powers, with the +50 D lens having no apparent effect on eye growth and refraction and the +65 D lens inducing myopia instead of hyperopia. CONCLUSIONS: Together these results argue that some sharp vision is fundamental to compensation to impose myopia. The significance of this new finding in relation to the processes underlying active emmetropization is discussed.

Biophysical properties of Na(v) 1.8/Na(v) 1.2 chimeras and inhibition by µO-conotoxin MrVIB.

BACKGROUND AND PURPOSE: Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na(v) 1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na(v) α-subtypes in neurons, Na(v) 1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na(v) 1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na(v) 1.8 and the TTX-S Na(v) 1.2 using a domain exchange strategy. EXPERIMENTAL APPROACH: Wild-type and chimeric Na(v) channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique. KEY RESULTS: MrVIB (1 µM) reduced Na(v) 1.2 current amplitude to 69 ± 12%, whereas Na(v) 1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na(v) 1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na(v) 1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na(v) 1.2. CONCLUSIONS AND IMPLICATIONS: Taken together, these results suggest that domain II of Na(v) 1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.

Affinity profiles of novel delta-receptor selective benzofuran derivatives of non-peptide opioids.

Highly selective heterocyclic opioid ligands with potent delta-antagonist activity have been developed on the basis of the "message-address" concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the mu-opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the delta-opioid receptor, much less potency toward the mu-binding site, and were the least effective at the kappa-site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers delta-receptor selectivity. The Na+ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dose-dependent.