Oxytocin-induced desensitization of the oxytocin receptor,☆☆

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Abstract

Objective: The purpose of this study was to characterize the oxytocin-induced desensitization of oxytocin-stimulated rises of intracellular calcium in cultured human myocytes. Study design: Culture lines were begun from biopsy specimens of myometrium that had been obtained from women who underwent low transverse cesarean deliveries. Fluorescence changes of calcium green-1 were used to demonstrate the rises of intracellular free calcium. Cells were exposed to 10 nmol/L oxytocin for 1 to 6 hours before the experimentation, allowed to rest for 10 minutes, and then tested for the fluorescence increases that resulted from exposure to 10 nmol/L oxytocin and μmol/L prostaglandin F2α. Subpopulations were defined as type 1 (responded to both oxytocin and prostaglandin F2α), type 2 (responded only to oxytocin), type 3 (responded only to prostaglandin F2α), or type 4 (responded to neither). The distribution of the subpopulations of cells was assessed by the determination of the response of every cell in every experimental run. Results: Pretreatment with oxytocin resulted in a decrease in the percentage of cells that responded to subsequent oxytocin exposure. The decrease was dependent on the duration of oxytocin exposure and was well fit with the Boltzmann sigmoid function. The duration of oxytocin exposure that yielded half-inactivation was 4.2 hours. Without oxytocin pretreatment, the distribution of subpopulations were 37.0% ± 18.0% (type 1), 23.1% ± 11.5% (type 2), 12.6% ± 8.0% (type 3), and 27.3% ± 22.9% (type 4). After 6 hours of oxytocin pretreatment, the percentage of type 1 and type 2 cells decreased to 2.4% ± 3.8% and 2.6% ± 2.4%, respectively, although the percentage of type 3 and type 4 cells increased to 20.4% ± 18.9% and 74.6% ± 22.1%, respectively. Conclusion: Oxytocin-induced desensitization of myocytes to oxytocin stimulation occurred over a clinically relevant time frame (4.2 hours). Continued responsiveness of the cells to prostaglandin F2α stimulation after 6 hours of oxytocin pretreatment indicated that postreceptor signaling pathways were maintained, which indicates that the oxytocin receptor likely is involved in the mechanism of myocyte desensitization to oxytocin stimulation. (Am J Obstet Gynecol 2003;188:497-502.)

Section snippets

Cell preparation

Human myometrium was obtained (according to the informed consent procedures that were approved by our institutional committee on human research) from the upper margin of the uterine incision at the time of cesarean delivery. Tissue was handled and dispersed into individual cells in a manner that was published previously.9 Briefly, an initial collagenase digestion was performed to loosen the cells from the connective tissue matrix, and a subsequent digestion was performed to obtain free cells.

Results

Fluorescence experiments that used the calcium-sensitive dye calcium green-1 were performed with a ×40 objective. An average of 50 cells (range, 35-65 cells) were observed for each trial. To determine the percentage of the cells that responded to a given stimuli, every cell in the field was analyzed for rises of intracellular calcium.

Fig 1 shows the experimental protocol and examples of the four types of cells that were observed.

. Raw fluorescence data show the four types of cells. Experimental

Comment

Understanding factors that control the responsiveness of myometrial cells to oxytocin is clearly important in modern obstetrics. The data we present here are the first to quantitate the loss of the ability of oxytocin to induce intracellular calcium rises as a function of oxytocin pretreatment. We also use an analysis of the distribution of subpopulations of myocytes to yield insight to the mechanism of oxytocin-induced desensitization.

These experiments were performed on cultured cells for

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Supported by the National Institutes of Health, Child Health and Human Development grant No. 1RO1HD 36373.

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Reprint requests: Roger C. Young, MD, PhD, Department of Obstetrics and Gynecology, One Medical Center Dr, Lebanon, NH 03756.

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