Differences were assessed as significant when < 0

Differences were assessed as significant when < 0.05. Open in a separate window Figure 4 Antagonism of ATP-stimulated ethidium accumulation in cells expressing the dog P2X7 receptor. long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. Oaz1 ATP possessed low millimolar potency at doggie P2X7 receptors. 2-&3-O-(4benzoylbenzoyl) ATP experienced slightly higher potency but Phenoxybenzamine hydrochloride was a partial agonist. Doggie P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies around the recombinant receptor although complete potency was considerably lower. Conclusions and implications: Doggie recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic. (2008). The dog P2X7 receptor was cloned from heart cDNA template using Phenoxybenzamine hydrochloride standard methods. Briefly, the dog P2X7 receptor, including the 5- and 3-un-translated regions, was amplified from dog heart cDNA by nested PCR using Pfu Turbo HotStart (Stratagene, La Jolla, CA, USA). The dog P2X7 receptor coding sequence obtained was confirmed from four templates (brain, heart and two different testis and ovary cDNA templates). The 1792 basepair product was then cloned into pENTR/D-TOPO (Invitrogen, LaJolla, CA, USA) to obtain the plasmid pENTR/D-DogP2X7 used for expression of the receptor. Construction of pFastBac-Mam-1 expression plasmids and BacMam-expression viruses The dog P2X7 receptor cDNA was subcloned into the BacMam baculovirus transfer vector, pFastBac-Mam-1, and BacMam baculovirus stocks were generated. Briefly, dog P2X7 cDNA was subcloned as a 1813 basepair Not1-to-Asc1 fragment from pENTR/D-DogP2X7 into the Not1 and Asc1 sites of pFastBac-Mam-NotAsc, which is a derivative vector of pFastBac-Mam-1 in which the polylinker region has been replaced by unique Not1 and Asc1 sites alone. The BacMam baculovirus transfer vector pFastBac-Mam-1 has been previously described (Condreay serotype 7136, Sigma, St. Louis, MO) at 37C. Thereafter, 50 L aliquots of blood were added to each well of a 96-well plate together with 30 L of phosphate-buffered saline or antagonist and the plates incubated for 40 min at 37C before adding 20 L of ATP. The plates were mixed and the mixtures incubated at 37C for 30 min (antagonist studies) or 0C100 min (agonist time course studies). Reactions were terminated by the addition of ice cold RPMI-1640 HEPES buffer (Invitrogen). The 96-well plates were centrifuged at 250for 5 min, and the resulting supernatants were harvested, diluted and their IL-1 content determined using a bioassay described previously (Buell test. Differences were assessed as significant when < 0.05. Open in a separate window Figure 4 Antagonism of ATP-stimulated ethidium accumulation in cells expressing the dog P2X7 receptor. HEK293 cells expressing the dog P2X7 receptor were pre-incubated for 40 min with antagonist before measuring ATP-induced ethidium accumulation. Studies were performed in NaCl buffer. Antagonists were pre-incubated with cells for 40 min before measuring ATP responses. (A) The effect of 1-(N,O-bis-[5-isoquinoline-sulphonyl]-N-methyl-L-tyroyl)-4-phenyl-piperazine (KN62) on ATP responses. (B) Transposition of the data in (A) to illustrate the effect of KN62 on responses to ATP. (C) The effect of brilliant blue G (BBG) on ATP responses. (D) Transposition of the data in (C) to illustrate the effect of BBG on responses to ATP. Basal ethidium accumulation in the absence of agonist is indicated on the X-ordinate as C in (A and C). The data are the mean SEM of 3C4 separate experiments. Open in a separate window Figure 5 Antagonism of ATP-stimulated ethidium accumulation in cells expressing the dog P2X7 receptor. HEK293 cells expressing the dog P2X7 receptor were pre-incubated for 40 min with Phenoxybenzamine hydrochloride antagonist before measuring agonist-induced ethidium accumulation. Studies were performed in NaCl buffer. Antagonists were pre-incubated with cells for 40 min before measuring ATP responses. (A) The effect of > 0.05, Dunnett’s test) although we could not directly compare maximal effects to ATP and BzATP in the same cells due to the methods used. Table 2 Effect of ATP and 2- & 3-O-(4benzoylbenzoyl) Phenoxybenzamine hydrochloride ATP (BzATP) at rat, human and dog P2X7 receptors in electrophysiological studies < 0.05) from value at rat P2X7 receptor but not significantly different to value at dog P2X7 receptor..