Beta-adrenoceptor subtype expression and function in rat white adipocytes.

1. The pharmacological features of rat white adipocyte beta-adrenoceptor subtypes were investigated by saturation and beta-agonist competition studies with [3H]-CGP 12177 and by lipolysis induced by beta-agonists as well as their inhibition by CGP 20712A (selective beta 1-antagonist) and ICI 118551 (selective beta 2-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of beta-adrenoceptor subtypes. 2. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22 +/- 0.07 nM) and the other with low affinity (23 +/- 7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3. The competition curves, with 15 nM [3H]-CGP 12177, for the beta-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective beta 3-agonist, BRL 37344 (BRL), were clearly biphasic (P < 0.001). The rank orders of agonist potency (pKi) in competing for [3H]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28 +/- 0.24) > NA (8.90 +/- 0.12) > Ad (8.65 +/- 0.12) > > BRL (4.53 +/- 0.17) and BRL (7.38 +/- 0.19) > > Iso (2.96 +/- 0.26) > or = NA (2.80 +/- 0.17) > Ad (2.10 +/- 0.11) indicating the expression of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective beta 1-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81 +/- 9 fmol mg-1) and high pKi value (7.23 +/- 0.26) confirming the presence of beta 1-adrenoceptors. 4. To assess a possible contribution of the beta 2-subtype, procaterol (Proc), a selective beta 2-agonist, was used to compete with 2 nM [3H]-CGP 12177. A single low affinity (4.61 +/- 0.07) population of binding sites was identified. The density of these sites (71 +/- 12 fmol mg-1) was similar to the one obtained with Xam, suggesting that Proc displaced [3H]-CGP 12177 from the beta 1-subtype. 5. The functional potency (pD2) order with BRL (9.07 +/- 0.20) and catecholamines (Iso: 7.26 +/- 0.06, NA: 6.89 +/- 0.02 and Ad: 6.32 +/- 0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31 +/- 0.06 and 5.66 +/- 0.10, respectively. Proc stimulated lipolysis with a low potency (5.59 +/- 0.21). 6. The lipolytic response to 0.001 microM BRL was inhibited by both, selective beta 1- and beta 2-antagonist, in a monophasic manner with low potencies (CGP 20712A pKi: < 4.5 and ICI 118551 pKi: 5.57 +/- 0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (> 10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 microM Iso or 0.13 microM NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pKi values for the first sites were 8.41 +/- 0.09 and 8.58 +/- 0.17, respectively, and for the second population of sites 4.73 +/- 0.22 and 4.27 +/- 0.27, respectively. The proportion of the first sites was low: 19 +/- 4 and 22 +/- 5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 microM Proc (CGP 20712A: pKi1: 8.17 +/- 0.08, site1: 23 +/- 6%, pKi2: 4.77 +/- 0.14; ICI 118551: pKi1: 7.78 +/- 0.03, site1: 37 +/- 2%, pKi2: 5.35 +/- 0.25). 7. Our results show that the radioligand [3H]-CGP 12177 allows the characterization of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on beta 1- and beta 3-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the beta 3-subtype.

Testosterone inhibits the basal and gonadotropin-releasing hormone-stimulated synthesis and release of newly synthesized alpha- and lutropin (LH) beta-subunit but not release of stored LH in cultured rat pituitary cells.

To further explore the mechanism of steroid feedback in male, the effects of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the rates of alpha- and lutropin (LH)beta-chain synthesis, neosynthesized subunits and radioimmunoassayable LH release into the medium were studied in the cultures of anterior pituitary cells from orchiectomized and intact rats. Polypeptides were [35S]methionine-labeled, immunoprecipitated separately in the medium and cells, then after SDS-PAGE precisely quantified. The total (medium + cells) radioactivity incorporated in the absence of GnRH into alpha- and LH beta-subunit was increased in orchiectomized rat cells vs. intact rat cells. GnRH stimulated the synthesis of both subunits, whether cells were from normal or castrated rat. T suppressed basal and GnRH-enhanced synthesis of both subunits in castrated rat cells. The values became closed to those observed in the normal rat cells. Also release of neosynthesized subunits from castrated rat cells into the culture medium was inhibited by T. In contrast, T did not change the basal and GnRH-induced radioimmunoassayed LH release. These results show that T can inhibit directly, at the pituitary level, alpha- and LH beta-subunit synthesis and neosynthesized but not stored LH release. They could explain, at least in part, no correlation between modifications of GnRH and LH secretion observed in vivo in response to T replacement.

Alteration of prostaglandin E receptors in advanced breast tumour cell lines.

We studied the direct effects of PGE2, often produced at high levels by mammary tumours, on three human breast cancer cell lines diversely advanced in malignancy regarding differentiation and tumorigenicity in nude mice. We evaluated PGE2 effect on cell growth, PGE2 receptor level and functionality. Our results show that PGE2 induces cAMP accumulation and inhibits the growth of the most differentiated breast cancer cells. We also demonstrate that loss and probably dysfunction of PGE2 receptors is related to an advanced tumorigenic phenotype of the cells. Thus, it seems that during progression of breast cancer, the cell growth escapes from control by PGE2. Nevertheless, it is possible to control the growth of advanced breast cancer cells in vitro by direct induction of intracellular cAMP accumulation.

Evidence for separate mechanisms of antiproliferative action of indomethacin and prostaglandin on MCF-7 breast cancer cells.

Indomethacin is reported to decrease the growth of many tumour cell lines. It is also known to be an anti-inflammatory agent acting by the inhibition of prostaglandin (PG) synthesis. To evaluate a clinical application as antitumoral agent, we have studied whether antiproliferative effect of indomethacin in breast cancer is related to its action on the prostaglandin production. We have observed that indomethacin as well as PGE1, PGE2, PGD2, and PGI2 inhibited the proliferation of MCF-7 breast cancer cells. As breast carcinomas were described to secrete mainly PGE2, we studied the effect of PGE2 on MCF-7 cells. These cells contain two types of binding sites for PGE2: high-affinity (Kd = 0.2 nM) and low-affinity (Kd = 20 nM) receptors. In this cell line, indomethacin and PGE2 inhibitory effects were additive. In addition, we showed that PGE2 increased the cAMP level in MCF-7 cells 30-fold (p < 0.001) while indomethacin did not change basal cAMP accumulation. Like for combination PGE2/indomethacin, the inhibitory effects of a cAMP analog (8-Br-cAMP) and indomethacin were additive. In conclusion, indomethacin inhibits the MCF-7 growth in specific manner independently of PG synthesis, PG action and cAMP accumulation.

Proliferative responses of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast pathogenesis.

We have explored the relationship of changes in proliferative responses of human mammary epithelial cells to a phorbol ester (TPA) and to 8-Br-cAMP, which modulate the activities of protein kinases A and C (PKA and PKC), with breast tumour progression. Treatment with TPA had no effect on nontumorigenic cell lines established from human fibrocystic biopsies and apparently normal tissue around a tumour. In contrast, TPA strongly inhibited the proliferation of numerous human tumorigenic breast cell lines. Treatment with 8-Br-cAMP decreased the proliferation of all studied nontumorigenic and tumorigenic cell lines. We have also studied the effect of TPA and 8-Br-cAMP on growth of epithelial cells in short-term culture obtained from surgical human mammary biopsies with different states of breast disease. Both drugs enhanced growth of normal breast cells but had no significant effects on cells from biopsies with benign breast disease. In contrast, all examined cultures from breast cancer biopsies were strongly inhibited by 8-Br-cAMP. Otherwise, TPA had an inhibitory effect only in the case of invasive ductal carcinoma of grade III. Malignant Ha-ras-transformation of nontumorigenic TPA-insensitive breast HBL-100 cells induced an inhibitory effect of TPA. In addition, a TPA-insensitive MCF7 clone was much less tumorigenic in athymic mice than the parental strain shown to be inhibited by TPA. These data suggest that the two intracellular transduction pathways change at different stages of breast pathogenesis. Alterations in the PKA pathway are early events and are probably important to cell immortalization but do not necessarily lead to malignant development. In contrast, changes in PKC pathway are rather later events associated with advanced malignant transformation.