OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidinium thiocyanate-phenol-chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde-1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase-polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase-polymerase chain reaction studies have confirmed expression of the phospholipase C-beta1a, phospholipase C-beta3, phospholipase C-gamma1, phospholipase C-beta2, and phospholipase C-delta1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-delta1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-beta1 a isoform was expressed at the highest level, the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-gamma2 isoforms at an intermediate level, and the phospholipase C-delta1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle.
OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidiniumthiocyanate-phenol-chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde-1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase-polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase-polymerase chain reaction studies have confirmed expression of the phospholipase C-beta1a, phospholipase C-beta3, phospholipase C-gamma1, phospholipase C-beta2, and phospholipase C-delta1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-delta1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-beta1 a isoform was expressed at the highest level, the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-gamma2 isoforms at an intermediate level, and the phospholipase C-delta1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle.