Ligand requirements for involvement of PKCε in synergistic analgesic interactions between spinal µ and δ opioid receptors.

BACKGROUND AND PURPOSE: We recently found that PKCε was required for spinal analgesic synergy between two GPCRs, δ opioid receptors and α2 A adrenoceptors, co-located in the same cellular subpopulation. We sought to determine if co-delivery of µ and δ opioid receptor agonists would similarly result in synergy requiring PKCε. EXPERIMENTAL APPROACH: Combinations of µ and δ opioid receptor agonists were co-administered intrathecally by direct lumbar puncture to PKCε-wild-type (PKCε-WT) and -knockout (PKCε-KO) mice. Antinociception was assessed using the hot-water tail-flick assay. Drug interactions were evaluated by isobolographic analysis. KEY RESULTS: All agonists produced comparable antinociception in both PKCε-WT and PKCε-KO mice. Of 19 agonist combinations that produced analgesic synergy, only 3 required PKCε for a synergistic interaction. In these three combinations, one of the agonists was morphine, although not all combinations involving morphine required PKCε. Morphine + deltorphin II and morphine + deltorphin I required PKCε for synergy, whereas a similar combination, morphine + deltorphin, did not. Additionally, morphine + oxymorphindole required PKCε for synergy, whereas a similar combination, morphine + oxycodindole, did not. CONCLUSIONS AND IMPLICATIONS: We discovered biased agonism for a specific signalling pathway at the level of spinally co-delivered opioid agonists. As the bias is only revealed by an appropriate ligand combination and cannot be accounted for by a single drug, it is likely that the receptors these agonists act on are interacting with each other. Our results support the existence of µ and δ opioid receptor heteromers at the spinal level in vivo. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Analgesic synergy between opioid and α2 -adrenoceptors.

UNLABELLED: Opioid and α2 -adrenoceptor agonists are potent analgesic drugs and their analgesic effects can synergize when co-administered. These supra-additive interactions are potentially beneficial clinically; by increasing efficacy and/or reducing the total drug required to produce sufficient pain relief, undesired side effects can be minimized. However, combination therapies of opioids and α2 -adrenoceptor agonists remain underutilized clinically, in spite of a large body of preclinical evidence describing their synergistic interaction. One possible obstacle to the translation of preclinical findings to clinical applications is a lack of understanding of the mechanisms underlying the synergistic interactions between these two drug classes. In this review, we provide a detailed overview of the interactions between different opioid and α2 -adrenoceptor agonist combinations in preclinical studies. These studies have identified the spinal cord as an important site of action of synergistic interactions, provided insights into which receptors mediate these interactions and explored downstream signalling events enabling synergy. It is now well documented that the activation of both µ and δ opioid receptors can produce synergy with α2 -adrenoceptor agonists and that α2 -adrenoceptor agonists can mediate synergy through either the α2A or the α2C adrenoceptor subtypes. Current hypotheses surrounding the cellular mechanisms mediating opioid-adrenoceptor synergy, including PKC signalling and receptor oligomerization, and the evidence supporting them are presented. Finally, the implications of these findings for clinical applications and drug discovery are discussed. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Visualization of spinal afferent innervation in the mouse colon by AAV8-mediated GFP expression.

BACKGROUND: Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno-associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV-driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. METHODS: Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. KEY RESULTS: Analysis of colon from rAAV8-GFP-treated mice demonstrated GFP-immunoreactivity (GFP-ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP-ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular-like clusters surrounding nuclei within myenteric ganglia. In addition, GFP-positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP-ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. CONCLUSIONS & INFERENCES: These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.

Laboratory evaluation of products to reduce settling of sweetpotato whitefly adults.

The impact of trademarked and commercial products on settling of adults of the sweetpotato whitefly, Bemisia tabaci (Gennadius), was studied in the laboratory. A no-choice bioassay using leaf disks of tomato, Solanum esculentum L., was developed to evaluate the impact of concentration series of products on settling of B. tabaci adults. The concentration of each product that would reduce settling by 50% (SC50) was estimated for each product using standard probit analyses, and the values were compared with that of Ultra-Fine Oil, a paraffinic oil product that is known to reduce settling of whitefly adults. Twenty-two trademarked products and 42 other products were evaluated in the laboratory bioassay. Based upon comparisons of fiducial limits of the respective SC50 values, Dawn detergent and E-RASE jojoba oil were the only trademarked products that were as effective as Ultra-Fine Oil in reducing settling of B. tabaci adults. Of the nontrademarked products, 25 were similar to Ultra-Fine Oil, although cedar, geranium, ginger, Hamlin (citrus), patchouli, olive and wintergreen oils, as well as citronellal and limonene, had ratios of respective SC50 values with that of Ultra-Fine Oil of approximately 1.5 or less. Combinations of limonene and citronellal with either olive oil or Ultra-Fine Oil were 15 and 30 times, respectively, more effective than Ultra-Fine Oil alone. Candidate products and combinations of products were further evaluated on tomato seedlings in no-choice screenhouse trials for effects on oviposition and on transmission of Tomato yellow leaf curl virus (family Geminiviridae, genus Begomovirus, TYLCV) by B. tabaci. Ultra-Fine Oil and olive oil reduced oviposition and transmission of TYLCV in the screenhouse trials. Ginger oil and limonene reduced oviposition in at least one screenhouse trial but did reduce transmission of TYLCV. The laboratory bioassay provided a rapid and relatively easy method to compare products for reducing settling of B. tabaci adults. Even though the reduced settling indicated in the laboratory bioassays was not always reflected in reduced oviposition or TYLCV transmission in the screenhouse trials, the bioassay was useful in rapidly identifying products that reduce settling and that could be investigated further.

Artificial substrates for oviposition and larval development of the pepper weevil (Coleoptera: Curculionidae).

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is a major pest of cultivated peppers (Capsicum spp.) and other cultivated and wild species within the family Solanaceae. Laboratory study of this insect, as well as its biological control agents, will be greatly facilitated by an artificial rearing system that does not rely on pepper fruit. An egg collection method and amendments to a standard larval diet were investigated for use in the rearing of this weevil. Spherical sachets made of Parafilm or netting enclosing leaves of pepper, American black nightshade, eggplant, tomato, potato, and jasmine tobacco induced oviposition. Tomato, potato, and jasmine tobacco leaves were accepted despite the fact that these are not oviposition hosts for pepper weevils in the wild. A standard larval diet formula was modified in an attempt to improve egg hatch, larval survival, developmental time, and adult mass. The diet formula was modified with the addition of freeze-dried jalapeño pepper powder, an additional lipid source, alternate protein sources, and the removal of methyl paraben. None of the aforementioned treatments resulted in a significant improvement over the standard diet. Egg hatch was greater when eggs were incubated on moist paper towels rather than in diet; thus, placement of neonates rather than eggs into diet improved production of adults. Suggestions for more efficient rearing of weevils on the currently available diet and future directions for the development of an artificial rearing system for pepper weevil are discussed.

Action threshold for applying insect growth regulators to tomato for management of irregular ripening caused by Bemisia argentifolii (Homoptera: Aleyrodidae).

The whitefly Bemisia argentifolii Bellows & Perring is a major pest of tomatoes, causing an irregular ripening disorder characterized externally by incomplete or inhibited reddening of fruit, especially in longitudinal sections, and internally by an increase in the amount of white tissue. Experiments were undertaken during the spring and fall of 1997 and 1998 and the spring of 1999 to develop an action threshold for applying the insect growth regulators (IGRs) buprofezin and pyriproxyfen to manage B. argentifolii and irregular ripening. The IGRs were applied when predetermined thresholds were reached and were compared with a high rate of the systemic insecticide imidacloprid, which was applied at transplanting and provided season-long whitefly control. Only plots treated when the numbers of sessile nymphs (second through fourth instars) reached five per 10 leaflets consistently had both external and internal irregular ripening severity ratings similar to the imidacloprid standard. Results were similar for buprofezin and pyriproxyfen even though the modes of action differ. The five nymphs per 10 leaflets threshold lends itself to field scouting because nymphal counts completed in the field using the unaided eye supplemented with a 10x hand lens were linearly and significantly related to counts completed in the laboratory with a dissecting microscope.

Aggregation pheromone for the pepper weevil,Anthonomus eugenii cano (Coleoptera: Curculionidae): Identification and field activity.

This study describes the identification of an aggregation pheromone for the pepper weevil,Anthonomus eugenii and field trials of a synthetic pheromone blend. Volatile collections and gas chromatography revealed the presence of six male-specific compounds. These compounds were identified using chromatographic and spectral techniques as: (Z)-2-(3,3-dimethylcyclohexylidene)ethanol, (E)-2-(3,3-dimethylcyclohexylidene)ethanol, (Z)-(3,3-dimethylcyclohexylidene)acetaldehyde, (E)-(3,3-dimethylcyclohexylidene)acetaldehyde, (E)-3,7-dimethyl-2,6-octadienoic acid (geranic acid), and (E)-3,7-dimethyl-2,6-octadien-1-ol (geraniol). The emission rates of these compounds from feeding males were determined to be about: 7.2, 4.8, 0.45, 0.30, 2.0, and 0.30µg/male/day, respectively. Sticky traps baited with a synthetic blend of these compounds captured more pepper weevils (both sexes) than did unbaited control traps or pheromone-baited boll weevil traps. Commercial and laboratory formulations of the synthetic pheromone were both attractive. However, the commercial formulation did not release geranic acid properly, and geranic acid is necessary for full activity. The pheromones of the pepper weevil and the boll weevil are compared. Improvements for increasing trap efficiency and possible uses for the pepper weevil pheromone are discussed. A convenient method for purifying geranic acid is also described.