Cloning, expression and tissue distribution of rat platelet‐activating‐factor‐receptor cDNA

Abstract

The biological functions of platelet-activating factor (PAF) have been extensively studied in the rat. However, the precise structure and distribution of rat PAF receptor has not been reported. To address this question, we isolated a rat PAF-receptor cDNA from a size-fractionated rat spleen cDNA library. The deduced amino acid sequence of the rat PAF receptor showed 80% and 79% identity with guinea pig and human PAF receptors, respectively. Pharmacological properties (ED,,, inhibition by WEB2086) of rat PAF receptors expressed in Xenopus oocytes were similar to those for PAF receptors expressed from guinea pig or human cDNAs. Northern blot analysis showed a widespread distribution of PAF-receptor mRNA in almost all organs including spleen, small intestine, kidney, lung, liver and brain. Considerable difference in the PAF-receptor distribution detected among species suggests the existence of a species-specific and tissue-specific regulatory mechanism for PAF-receptor-mRNA expression. Isolation of rat PAF-receptor cDNA should facilitate further analysis of PAF-receptor function and pharmacology in diverse pathophysiological processes.

Regulation of platelet-activating factor receptor gene expression in vivo by endotoxin, platelet-activating factor and endogenous tumour necrosis factor

“…Genomic DNA, a possible impurity in the RNA extract, was not considered an important source of interference. This is because (i) the 5h-1\3h-3 primer pair walks through a large intron [21], and (ii) this large intron could not be amplified by PCR under the conditions of our experiment, as reported by Bito et al [21].…”

“…A CAAATTCC sequence exists in the cDNA of rat PAF-R from bp 258 to 265 [21]. The adenine at bp 259 (underlined) could be changed to guanine using a site-specific mutagenesis PCR [27], resulting in a cDNA fragment containing a mutated CGAATTCC sequence, which can be digested with EcoRI restriction enzyme.…”

“…Studies on comparative quantification of PAF-R in organ\tissue are rare, probably because of the technical difficulty of differentiating membrane binding and membrane intercalation of a phospholipid [2,17]. The recent development of quantitative PCR and cloning of PAF-R cDNA from guinea-pig [18], human [19,20] and rat [21] tissues has greatly facilitated our understanding of PAF-R gene expression and regulation. Recent studies in itro on the regulation of PAF-R gene transcription by inflammatory stimuli Abbreviations used : PAF, platelet-activating factor ; PAF-R, PAF receptor ; LPS, lipopolysaccharide ; TNF, tumour necrosis factor α ; RT-PCR, reverse transcriptase-PCR ; Q-PCR, quantitative RT-PCR ; cRNA, competitor RNA ; WEB, PAF antagonist ; WBC, white blood cell count ; PMN, polymorphonuclear leucocyte ; i.p., intraperitoneal ; i.v., intravenous.…”

Increased hepatic platelet activating factor (PAF) and PAF receptors in carbon tetrachloride induced liver cirrhosis

“…34 35 The reaction was terminated by addition of 5 ml of ice cold assay buffer, filtration through presoaked Whatman GF/C filters (Whatman, Hillsboro, Oregon, USA), washed thrice, and radioactivity was determined in a b scintillation counter.…”

“…29 35 The reaction for PAF was carried out as follows: denaturation at 94˚C (one minute), annealing at 60˚C (30 seconds), and extension at 72˚C (30 seconds) for 35 cycles 35 as linearity was observed between 20 and 45 cycles. Reaction for b-actin (for normalisation) was carried out for 30 cycles, essentially as described previously.…”