Comparative Studies on the In Vitro: RESULTS(5)
Short-Term dbcAMP Treatment
Human and baboon stromal cells were pretreated with hormones for 2, 12, or 17 days, and dbcAMP was added for the last 48 h of each time point. This was done in order to determine whether long- or short-term treatment with dbcAMP was required for decidualization and IGFBP-1 expression. In human stromal cells, levels of IGFBP-1 significantly increased in response to 48 h of dbcAMP treatment at both 2 and 12 days (Fig. 5B; p < 0.01). In baboon cells, a significant increase in IGFBP-1 was observed in response to dbcAMP only after 17 days of hormone pretreatment (Fig. 5A; p < 0.01). The quantity of IGFBP-1 produced by both human and baboon cells in response to short-term dbcAMP treatment (Fig. 5) was much lower than the levels obtained after long-term treatment with dbcAMP (Fig. 3). These data show that dbcAMP is able to act in a short-term manner to induce IGFBP-1 production when cells are pretreated with hormones. Furthermore, it is evident that cells at the initial stages of hormone treatment differ in their responsiveness to dbcAMP from cells subjected to long-term hormone pretreatment. buy levaquin online
PKA Regulatory Subunits
One mechanism by which the action of cAMP can be regulated is by the controlled expression of the various PKA regulatory subunits: RIa, RIp, RIIa, and RIIp. Human stromal cells expressed mRNAs for all four subunits (Fig. 6B). RIa was the major subunit expressed, followed by RIIa and RIIp, with the weakest expression being that of RIp. There was no observable difference in the expression of these subunits between decidualized and nondeci-dualized cells. In baboon stromal cells, RIIp was the major subunit expressed (Fig. 6A). RIa and RIIa were also expressed; however, no RIp was detected. Similar to the situation with human cells, no observable difference in expression was evident between decidualized and nondeci-dualized cells.
FIG. 5. Short-term dbcAMP treatment of endometrial stromal cells pretreated with hormones. Confluent stromal cells from baboon (A) and human (B) endometrium were treated with hormones for 2, 12, or 17 days. At 48 h prior to each sampling time point, fresh medium supplemented with 0.1 mM dbcAMP was added to cells. IGFBP-1 in the culture medium and cytosol extracts was measured by RIA. Data are expressed as least-squares means ± SEM of 3 baboon or 2 human tissues. * p s 0.01, significant difference compared to hormone treatment only.
FIG. 6. Expression of the various PKA regulatory subunits in decidualized cells. Messenger RNA isolated from baboon (A) and human (B) nondecidualized (no IGFBP-1 expression, lane 1) and decidualized cells (high IGFBP-1-expressing cells, lane 2) were subjected to RT-PCR using primers to the regulatory subunits RIa, RIp, RIIa, RIIp. The autoradiograms are representative experiments for 3 baboon and 3 human tissues. Values for the graphs were obtained by image analysis using the PhosphorImager system and expressed as a ratio of H3.3. Data are presented as least-squares means ± SEM of 3 baboon and 3 human tissues.