Multiple G proteins and phospholipase C isoforms in human myometrial cells: implication for oxytocin action.
Although a physiological role for oxytocin during parturition is well accepted, the mechanisms by which it activates myometrial contractility during labor have not been completely elucidated. We have previously shown the presence of Gq and two pertussis toxin (PT) substrates of the Gi family in human myometrial cells. In the present study, we have identified by Western blotting the G protein and phospholipase C (PLC) isoforms present in these cells and investigated their implication in oxytocin signaling by measuring the formation of inositol phosphates (IPs) and mobilization of intracellular calcium. We found G protein subunits alpha(q), alpha(11), alpha(i1), alpha(i2), alpha(i3), alpha(z), and two splice variants of alpha(s)- and beta-subunits. We have also detected the presence of five PLC isoforms: beta 1, beta 2, beta 3, gamma 1, and gamma 2. Oxytocin-induced IPs formation and intracellular Ca2+ mobilization were inhibited to approximately 50% after pretreatment of the cells with PT, suggesting that oxytocin activates PLC beta by interacting with at least two types of G proteins: a member of the Gq family (PT resistant) and a member of the Gi family (PT sensitive). The tyrosine phosphatase inhibitor pervanadate stimulated IPs formation in myometrial cells. Using the protein kinase inhibitors staurosporine, phenylarsine oxide, and Ro 31-8220 and the protein kinase C activator phorbol dibutyrate, we have shown that pervanadate and oxytocin activate PLC by different mechanisms. Furthermore, oxytocin did not activate tyrosine phosphorylation in human myometrial cells, as measured with an antiphosphotyrosine antibody, indicating that it does not activate a PLC gamma isoform. We conclude that oxytocin activates human myometrium by interacting with at least two G proteins and possibly three PLC beta isoforms.
Phaneuf
S
S
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women's Centre, John Radcliffe Hospital, United Kingdom. sphaneuf@immsvr.jr2.ox.ac.uk
Carrasco
M P
MP
Europe-Finner
G N
GN
Hamilton
C H
CH
López Bernal
A
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009215:Myometrium / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D010121:Oxytocin / Q000502:physiology*; D010455:Peptides / Q000276:immunology; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D007106:Immune Sera; D007527:Isoenzymes; D010455:Peptides; D010121:Oxytocin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium