β-Adrenergic receptor subtype gene expression in timed-pregnant rat myometrium

Abstract

OBJECTIVE: Classic radioligand binding techniques have suggested that β 1- and β 2-adrenergic receptor subtype proteins are expressed in myometrial tissue; however, to date these observations have not been confirmed at the level of the messenger ribonucleic acid for these clinically important membrane receptors. The studies described in this report sought to use quantitative reverse transcriptase–polymerase chain reaction techniques to confirm expression of messenger ribonucleic acid for the β 1- and β 2-adrenergic receptors in myometrial tissue and to determine whether messenger ribonucleic acid expression for these two adrenergic receptors is modulated during pregnancy. STUDY DESIGN: For these studies total cellular ribonucleic acid was isolated from myometrial tissue obtained from timed-pregnant Sprague-Dawley rats by the guanidium thiocyanate–phenol–chloroform extraction technique; formaldehyde-agarose gels then confirmed isolation of intact ribonucleic acid. Random hexamer primers and reverse transcriptase were used to synthesize complementary deoxyribonucleic acid. Subsequently, polymerase chain reaction was performed with subtype specific 20-mer sense and antisense oligonucleotide primers specific for the rat β 1- and β 2-adrenergic receptors. Inclusion of internal standard deoxyribonucleic acid sequences allowed quantification of the reverse transcriptase–polymerase chain reaction results. RESULTS: By use of total cellular ribonucleic acid isolated from myometrial tissue, reverse transcriptase–polymerase chain reaction generated the expected 328 bp product for the β 1-receptor and the expected 559 bp product for the β 2-receptor along with internal standard deoxyribonucleic acid sequences for both. The identity of the β 1- and β 2-adrenergic receptor polymerase chain reaction products was confirmed on the basis of restriction endonuclease digestions producing the expected deoxyribonucleic acid fragments and by Southern blots using β 1- and β 2-adrenergic receptor–specific complementary deoxyribonucleic acid probes. The reverse transcriptase–polymerase chain reaction studies confirmed a gradual decline in β l-receptor messenger ribonucleic acid and stable expression of β 2-receptor messenger ribonucleic acid during the second half of gestation in pregnant rat myometrial tissue. CONCLUSIONS: In summary, these studies have confirmed, at the messenger ribonucleic acid level, expression of the β 1- and β 2-adrenergic receptor subtypes in timed-pregnant rat myometrial tissue.(Am J Obstet Gynecol 1997;176:349-57.)