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Molecular cloning, expression and pharmacological characterization of the canine cholecystokinin 1 receptor.

ABSTRACT: 1 The full-length, canine cholecystokinin 1 (CCK1) receptor was cloned from gallbladder tissue using RT-PCR with a combination of primers designed to interact with conserved regions of the human and rat CCK1 receptor, which also shared homology with the canine genomic sequence. 2 Analysis of the sequence of the canine CCK1 receptor revealed a 1287 base pair product, which encoded a 429 amino-acid protein. This protein was 89% identical to the human and 85% identical to the rat CCK1 receptor. 3 The canine CCK1 receptor was expressed in CHO-K cells for pharmacological characterization. In competition studies, using [(125)I]BH-CCK-8S as radioligand, the affinity values estimated for CCK receptor-selective compounds were not significantly different between the canine and human CCK1 receptors (pK(I)+/-s.e.m. at canine CCK1 receptor; L-364,718=8.82+/-0.08, L-365,260=6.61+/-0.05, YF476=7.91+/-0.15, YM022=8.28+/-0.06 and dexloxiglumide=7.53+/-0.11). Furthermore, the selectivity of these compounds between canine CCK1 and CCK2 receptors was consistent with the selectivity between the human CCK1 and CCK2 receptors. 4 Two additional forms of the canine CCK1 receptor were identified during the cloning procedure. These had three (variant #1) and six (variant #2) amino-acid differences from the wild-type canine CCK1 receptor. Variant #1 bound [(125)I]BH-CCK-8S and displayed an identical pharmacological profile to the wild-type receptor using the ligands described above. No significant binding was measured with variant #2. 5 In conclusion, we have cloned and pharmacologically characterized the canine CCK1 receptor. The data obtained will facilitate the interpretation of numerous pharmacological experiments that have been performed using canine tissue to elucidate the actions of CCK and gastrin.

SUBMITTER: Morton MF

PROVIDER: S-EPMC1576148 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Molecular cloning, expression and pharmacological characterization of the canine cholecystokinin 1 receptor.

Morton Magda Francesca MF Pyati Jayashree J Dai Heng H Li Lina L Moreno Veronica V Shankley Nigel Paul NP

British journal of pharmacology 20050601 3

1 The full-length, canine cholecystokinin 1 (CCK1) receptor was cloned from gallbladder tissue using RT-PCR with a combination of primers designed to interact with conserved regions of the human and rat CCK1 receptor, which also shared homology with the canine genomic sequence. 2 Analysis of the sequence of the canine CCK1 receptor revealed a 1287 base pair product, which encoded a 429 amino-acid protein. This protein was 89% identical to the human and 85% identical to the rat CCK1 receptor. 3 T ...[more]

PMID: 15765098

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Project description:Mammalian beta-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine beta-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the beta-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine beta-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 mug/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 mug/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 mug/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 mug/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine beta-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine beta-defensins play an important role in host defense and other physiological functions of the male reproductive system.

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Molecular cloning, expression and pharmacological characterization of the canine cholecystokinin 1 receptor.

Publications

Molecular cloning, expression and pharmacological characterization of the canine cholecystokinin 1 receptor.

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