(S-284) Gogarten, W., Saturday 9:15
TITLE: CHRONIC PRETREATMENT WITH TUMOR NECROSIS FACTOR ALPHA (TNF) STIMULATES ADENYLYL CYCLASE ACTIVITY IN CULTURED HUMAN MYOMETRIAL CELLS
AUTHORS: Wiebke Gogarten, MD1, Karen S. Lindeman, MD2, Carol A. Hirshman, MD3, Charles W. Emala, MD3
AFFILIATION: 1Columbia Univ., New York, NY/Westfaelische Wilhelms-Universitaet, Muenster, Germany; 2Johns Hopkins Med. Institutions, Baltimore, MD; 3Columbia Univ., New York, NY.
INTRODUCTION: Intrauterine infection and cytokine release have been implicated as important causes of preterm labor, however, little is known about the underlying mechanism. Although some studies have shown increased levels of TNF and interleukin-6 during intrauterine infection (1), TNF and interleukin-6 increase cAMP levels in human myometrial cells (2). An important mechanism of uterine relaxation and quiescence during pregnancy is the activation of Gs-coupled receptors with stimulation of adenylyl cyclase, which in turn catalyzes the conversion of ATP to cAMP. We therefore investigated the effect of chronic TNF exposure on adenylyl cyclase activity in human myometrial cells.
METHODS: Cell cultures from human myometrium (passages 4-9) were grown to confluency, serum-starved, and chronically pretreated with TNF (10 ng/ml) for 12, 18, 24, or 48. Basal, GTP (10-5 M)-, NaF (10-2 M)-, forskolin (10-5 M)-, and MnCl2 (20 mM)-stimulated adenylyl cyclase activity was measured in triplicates in prepared membrane fractions for 20 min, after which the reaction was stopped. results are given as picomoles of cAMP/mg protein ± SEM. Statistical analysis were performed using paired t-test. A p-value < 0.05 was considered significant.
RESULTS: Chronic pretreatment with TNF led to an increase in basal, GTP-, NaF-, and forskolin-stimulated adenylyl cyclase activity after 48 h, while MnCl2-stimulated activity remained unchanged (Table 1).
|
|
Control |
TNF (48 h) |
p |
|
Basal |
88±32 |
184±29 |
0.01 |
|
GTP |
144±29 |
415±63 |
0.02 |
|
NaF/AlCl3 |
1164±182 |
2136±295 |
0.02 |
|
Forskolin |
1431±332 |
3102±425 |
0.01 |
|
MnC12 |
1521±167 |
2230±398 |
n.s. |
CONCLUSION: Increased adenylyl cyclase activity after chronic TNF exposure in human myometrial cells suggests an enhancement of uterine relaxation and further supports the notion that TNF is not involved in the induction of premature labor. The lack of effect of TNF on MnCl2-stimulated adenylyl cyclase activity and the time course suggest that the effects of TNF occur proximal to the adenylyl cyclase level (i.e., Gs alpha) and that long-term modifications requiring protein synthesis are involved.
REFERENCES:
1. Obstet Gynecol 1993;81:941-948.
2. Am J Reprod Immunol 1993;30:49-57.
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