Pharmacological and genetic characterisation of the canine P2X4 receptor.

BACKGROUND AND PURPOSE: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. EXPERIMENTAL APPROACH: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. KEY RESULTS: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2'(3')-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. CONCLUSION AND IMPLICATIONS: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans.

A case of monocephalusrachipagus tribrachius tetrapus in a puppy.

Caudal duplication (dipygus) is an uncommon pathologic of conjoined twinning. The conjoined malformation is classified according to the nature and site of the union. We report the presence of this malformation in a female crossbreed puppy. The puppy was delivered by caesarean section following a prolonged period of dystocia. External findings showed a single head (monocephalus) and a normal cranium with no fissure in the medial line detected. The thorax displayed a caudal duplication arising from the lumbosacral region (rachipagus). The puppy had three upper limbs, a right and left, and a third limb in the dorsal region where the bifurcation began. The subsequent caudal duplication appeared symmetrical. Necropsy revealed internal abnormalities consisting of a complete duplication of the urogenital system and a duplication of the large intestines arising from a bifurcation of the caudal ileum . Considering the morphophysiological description the malformation described would be classified as the first case in the dog of a monocephalusrachipagustribrachius tetrapus.

R270C polymorphism leads to loss of function of the canine P2X7 receptor.

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.

Functional expression of the damage-associated molecular pattern receptor P2X7 on canine kidney epithelial cells.

Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin-Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5'-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2'(3')-O-(4-benzoylbenzoyl)ATP induced ethidium(+) uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1ß and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans.

Activation of the damage-associated molecular pattern receptor P2X7 induces interleukin-1ß release from canine monocytes.

P2X7, a damage-associated molecular pattern receptor and adenosine 5'-triphosphate (ATP)-gated cation channel, plays an important role in the activation of the NALP3 inflammasome and subsequent release of interleukin (IL)-1ß from human monocytes; however its role in monocytes from other species including the dog remains poorly defined. This study investigated the role of P2X7 in canine monocytes, including its role in IL-1ß release. A fixed-time flow cytometric assay demonstrated that activation of P2X7 by extracellular ATP induces the uptake of the organic cation, YO-PRO-1(2+), into peripheral blood monocytes from various dog breeds, a process impaired by the specific P2X7 antagonist, A438079. Moreover, in five different breeds, relative P2X7 function in monocytes was about half that of peripheral blood T cells but similar to that of peripheral blood B cells. Reverse transcription-PCR demonstrated the presence of P2X7, NALP3, caspase-1 and IL-1ß in LPS-primed canine monocytes. Immunoblotting confirmed the presence of P2X7 in LPS-primed canine monocytes. Finally, extracellular ATP induced YO-PRO-1(2+) uptake into and IL-1ß release from these cells, with both processes impaired by A438079. These results demonstrate that P2X7 activation induces the uptake of organic cations into and the release of IL-1ß from canine monocytes. These findings indicate that P2X7 may play an important role in IL-1ß-dependent processes in dogs.

Permethrin spot-on intoxication of cats Literature review and survey of veterinary practitioners in Australia.

UNLABELLED: SURVEY AIMS: A questionnaire was sent to veterinarians in Australia to determine the approximate number of cats presenting for permethrin spot-on (PSO) intoxication over a 2-year period. FINDINGS: Of the 269 questionnaires returned, 255 were eligible for analysis. A total of 207 respondents (81%) reported cases of PSO intoxication in cats over the previous 2 years. In total, 750 individual cases were reported, with 166 deaths. While all deaths were generally attributable to intoxication, 39 cats were euthanased because owners were unable to pay the anticipated treatment costs. Brands of PSO implicated included Exelpet Flea (and Tick) Liquidator (Mars Australia) (146 respondents), Bayer Advantix (48), Purina Totalcare Flea Eliminator Line-On (19), Troy Ease-On (six) and Duogard Line-On (Virbac) (four); 67 respondents were not able to identify a specific product. Permethrin spot-on formulations were most commonly obtained from supermarkets (146 respondents), followed by pet stores (43), veterinary practices (16), and a range of other sources including produce stores and friends. The majority of intoxication cases reported involved PSOs labelled for use in dogs with specific label instructions such as 'toxic to cats'. Owners applied these PSO products to their cats accidentally or intentionally. In some cases, exposure was through secondary contact, such as when a PSO product was applied to a dog with which a cat had direct or indirect contact. RECOMMENDATIONS: In the authors' view, because of the likelihood of inappropriate use and toxicity in the non-labelled species, over-the-counter products intended for use in either dogs or cats must have a high margin of safety in all species. Furthermore, PSOs should only be available at points of sale where veterinary advice can be provided and appropriate warnings given. As an interim measure, modified labelling with more explicit warnings may reduce morbidity and mortality.

Monocephalus dipygus parapagus: a suspected case of complete caudal duplication in a British Blue kitten.

Complete caudal duplication is an extremely rare type of conjoined twinning. This communication reports the birth and gross features of a live conjoined British Blue kitten born naturally to a healthy queen. The monocephalic dipygus kitten exhibited a normal skull and upper body, two separate pelves, four hind limbs and two tails. A cleft palate and bifurcation of rostral mandible were also noted. The absence of maternal dystocia was unusual for conjoined twinnings.