Abstract
The contractile effect of okadaic acid (OA), a highly selective inhibitor of protein serine/threonine phosphatases, was analyzed in the rat uterus during the estrous cycle and during the course of pregnancy. Contractile effects were related to circulating levels of estrogen and progesterone and to mRNA levels of myosin light chain kinase (MLCK) and of myosin light chain protein phosphatase catalytic (PP1-δ) and larger regulatory subunit (MYPT). Both in nonpregnant and pregnant uteri, OA (20 μM) induced a transient contraction, which after plateauing, slowly decreased. In the nonpregnant uterus, the amplitude of this contraction varied at different stages of the estrous cycle, being higher at proestrus and lower at diestrus. In the pregnant uterus, the contraction to OA increased significantly during the course of pregnancy, reaching a maximum in day 21 pregnant rats, and declined after delivery. Whatever the day of pregnancy, the amplitude of the contraction to OA was not significantly modified when obtained in Ca2+-free solution. The magnitude of the OA-induced contraction in spontaneously cycling and pregnant rats was positively correlated to the ratio of estrogen/progesterone serum levels. Reverse transcription-polymerase chain reaction assays on myometrial tissue demonstrated that mRNA expression of PP1-δ and MYPT was higher at early (day 3) than at late (day 21) pregnancy. MLCK mRNA levels were similar in day 3 and day 21 pregnant rats. These data suggest that changes in the expression and activity of myosin phosphatase may contribute to modulating the level of uterine contractile force during the estrous cycle, pregnancy, and labor.
Footnotes
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Send reprint requests to: Dr. Francisco M. Pinto, Instituto de Investigaciones Quı́micas, Centro de Investigaciones Cientı́ficas Isla de La Cartuja, Avda. Americo Vespucio s/n, 41092 Sevilla, Spain. E-mail: mluz{at}cica.es
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This work was supported by grants from the Ministry of Education and Science (Spain) (PB 97-1123) and from Fundación Ramón Areces (Spain). M.T. is the recipient of a fellowship from Fundación Ramón Areces, Spain. C.G.C. is the recipient of a fellowship from the Ministry of Science, Spain.
- Abbreviations:
- [Ca2+]i
- cytosolic Ca2+ concentration
- MLCK
- myosin light chain kinase
- LC20
- 20-kDa myosin light chain
- MLCPP
- myosin light chain protein phosphatase
- PP1-δ
- protein phosphatase type 1 δ-isoform
- MYPT
- large myosin phosphatase target subunit
- OA
- okadaic acid
- PSP
- protein serine/threonine phosphatases
- E2
- 17β-estradiol
- P4
- progesterone
- RT-PCR
- reverse transcription-polymerase chain reaction
- PSS
- physiological salt solution
- ACh
- acetylcholine
- bp
- base pairs
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- Received August 1, 2000.
- Accepted October 31, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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