Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnant rat myometrial tissue.
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.


METHODS
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.
Bieber
E
E
Department of Obstetrics and Gynecology, University of Chicago, Illinois 60637, USA.
Stratman
T
T
Sanseverino
M
M
Sangueza
J
J
Phillippe
M
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D005260:Female; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics*; D009215:Myometrium / Q000201:enzymology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017207:Rats, Sprague-Dawley; D017385:Sequence Homology; D010738:Type C Phospholipases / Q000235:genetics*
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase