Expression of prostaglandin G/H synthase (PGHS) and heat shock protein-70 (HSP-70) in the corpus luteum (CL) of prostaglandin F2 alpha-treated immature superovulated rats.

In this study we examined the mechanism of corpus luteum (CL) regression by measuring changes in expression of prostaglandin G/H synthase-1 (PGHS-1) and -2 (PGHS-2) in day 4 CL and inducible heat shock protein 70 (HSP-70) in day 4 and day 9 CL of immature superovulated rats. The rats were superovulated and treated with 500 microg of prostaglandin F2alpha (PGF2alpha) on day 4 or day 9 after CL formation. Ovaries and serial blood samples were removed during the 24-hour period following treatment. Plasma progesterone was determined by radioimmunoassay while mRNA abundance and protein expression were assessed by semiquantitative RT-PCR and immunoblot analysis, respectively. One hour after PGF2alpha, both day 4 and day 9 rats exhibited a significant decrease in progesterone secretion; however, there was a greater decrease in day 9 rats. In ovarian samples removed on day 4, there was a significant increase in mRNA for PGHS-2 at 1 hour after PGF2alpha. PGHS-1 mRNA content remained unchanged. Immunoblot analyses showed an increase in PGHS-2 protein expression only at 8 h. There were no changes in PGHS-1 protein expression. In day 9 rats, ovarian HSP-70 protein levels increased by 50% after PGF2alpha injection; however, on day 4 there was no change in expression of this protein over the sampling period. These results suggest that expression of PGHS-2 may be involved in inhibiting progesterone production and that expression of HSP-70 may be required for complete CL regression in the rat.

Treatment with prostaglandin F2alpha increases expression of prostaglandin synthase-2 in the rat corpus luteum.

Recent studies indicate that the corpus luteum (CL) may be a source of prostaglandin F2alpha (PGF2alpha) for regression. We investigated expression of mRNA and protein for prostaglandin G/H synthase (PGHS) in the CL of immature superovulated rats following administration of PGF2alpha. We observed an increase in mRNA for PGHS-2, the induced isoform, at 1 h and protein at 8 and 24 h after treatment. One hour after PGF2alpha, there was also a progressive decrease in plasma progesterone concentration. There were no changes, however, in expression of PGHS-1, the constitutive isoform, over the 24 h sampling period. These results indicate that PGHS-2 increases following PGF2alpha treatment and that expression of this enzyme in the rat CL may contribute to the luteolytic mechanism.

Pharmacological and molecular biological (RT-PCR) characterization of functional TP prostanoid receptors in immortalized human non-pigmented ciliary epithelial cells.

Immortalized human non-pigmented ciliary epithelial (NPE) cells (ODM-2) were shown to express the mRNA for the prostanoid TPalpha but not the TPbeta receptor using reverse transcription-polymerase chain reaction (RT-PCR). These TPalpha receptors were coupled to phospholipase C (PLC) and, thus, promoted phosphoinositide (PI) turnover. TP receptor agonists yielded the following potencies (EC50S) in the PI turnover assays: I-BOP = 8.2 +/- 1.1 nM; carbocyclic TA2 = 87.5 +/- 25.3 nM; U-44069 = 1.16 +/- 0.32 microM; U-46619 = 1.2 +/- 0.2 microM (n = 4-17). Agonists selective for other prostanoid receptor subtypes (e.g., fluprostenol and sulprostone) were inactive. The agonist effects of U-44619 and I-BOP were potently blocked, in an apparent non-competitive manner (ki = 53.9 +/- 12 nM; pA2s = 7.6-7.8; pKbs = 7.38), by the TP receptor-selective antagonist, SQ29,548, but were unaffected by other prostanoid receptor antagonists (e.g., AH6809, AL-8810). The PLC inhibitor (U73122) inhibited U-46619-induced PI turnover (IC50 = 4.3 +/- 0.6 microM). The functional potencies of the compounds stimulating or inhibiting the TP receptor-mediated PI turnover in the NPE cells correlated well with the TP receptor binding affinities of these compounds at human platelet TP receptors (r = 0.98). These studies have shown the presence of the mRNA for and the expression of functional TPalpha receptors coupled to PLC in human NPE cells. The TPalpha receptors on NPE cells may be responsible for inhibiting aqueous humor production and may help explain the intraocular pressure-lowering effects of certain TP agonists.

Operational correlates of prostanoid TP receptor expression in human non-pregnant myometrium are unaffected by excision site or menstrual cycle status of the donor.

1. Cumulative concentration-effect curves for the selective prostanoid TP receptor agonist, U46619, were constructed in strips of human non-pregnant myometrium grouped according to tissue excision site (top, lateral wall, lower uterine segment, sub-serosal or sub-endometrial), tissue orientation (strips cut either parallel or perpendicular to the serosa) and donor menstrual status (proliferative or secretory phase). 2. U46619 was excitatory in all tissues. There was no significant difference in either pEC50 or maximum response between groups (P<0.05). The range of pEC50 values was 6.8+/-0.1 (lateral wall, proliferative phase, n=5) to 7.1+/-0.3 (lateral wall, secretory phase, n=5). The range of maximum response values was 0.9+/-0.8 N cm-2 (lateral wall, proliferative phase, n=5) to 3.1+/-1.0 N cm-2 (lateral wall, secretory phase, n=5). 3. Saturation binding analyses were conducted using the radiolabelled TP receptor agonist, [125I]-BOP. Binding parameters were estimated for membranes prepared from human non-pregnant myometrium excised from the lateral wall and grouped according to donor menstrual status. 4. There were no significant differences in the mean pKd and [R]tot values for [125I]-BOP binding between the two groups (proliferative phase: pKd=8.3+/-0.3, [R]tot=412+/-319 fmol mg protein-1, n=5; secretory phase: pKd=8.5+/-0.4, [R]tot=369+/-192 fmol mg protein-1, n=6; P<0.05). 5. These data indicate that U46619-mediated responses in human non-pregnant myometrium are not influenced by tissue excision site, tissue orientation or donor menstrual status and that [125I]-BOP binding is not influenced by donor menstrual status. This suggests that the TP receptor population is homogeneous throughout the human non-pregnant myometrium, and not subject to hormonal regulation.

Characterization of the prostanoid TP receptor population in human nonpregnant myometrium.

We have used both functional and binding studies to fully characterize the prostanoid TP receptor in the myometrium from nonpregnant human donors. Both U-46,619 and I-BOP produced concentration-dependent contraction of human myometrial strips in vitro (pEC50 = 6.9 +/- 0.6; and 7.8 +/- 0.5, respectively). U-46,619-induced contractions were attenuated by the TP receptor antagonists: ICI 192,605 (pKB = 9.2 +/- 0.3); ICI D1,542 (pKB = 9.1 +/- 0.3); L670,596 (pKB = 8.6 +/- 0.3); GR 32,191 (pKB = 8.6 +/- 0.2); SQ 29,548 (pKB = 8.2 +/- 0.5); ONO 3,708 (pKB = 8.1 +/- 0.3) and BM 13,505 (pKB = 7.4 +/- 0.2). The binding of [125I]-BOP to human myometrial membranes was saturable, selective and displaceable. Equilibrium binding of [125I]-BOP identified one class of sites, Kd = 3.4 nM (pKd = 8.7 +/- 0.4) and a maximum binding of 323.1 +/- 361.5 fmol/mg protein. The addition of the nonhydrolyzable GTP analog GTP gamma S (100 microM) to the assay had no effect on [125I]-BOP binding. The Kd determined kinetically was 4.1 +/- 0.2 nM. TP receptor antagonists competed for [125I]-BOP binding: ICI D1,542 (pIC50 = 8.3 +/- 0.4); L670,596 (pIC50 = 7.9 +/- 0.1); ICI 192,605 (pIC50 = 7.2 +/- 0.1); ONO 3,708 (pIC50 = 7.2 +/- 0.04); SQ 29,548 (pIC50 = 7.2 +/- 0.1); GR 32,191 (pIC50 = 7.0 +/- 0.2); BM 13,505 (pIC50 = 6.8 +/- 0.1). The rank order of potency for the seven TP receptor antagonists in displacing [125I]-BOP from its binding site was correlated (r = 0.75) with the rank order of potency in inhibiting U-46,619-induced contraction of myometrial strips. Ligands selective for other prostanoid receptors were unable to significantly displace [125I]-BOP binding. These results are consistent with the notion that the human myometrial TP receptor is pharmacologically similar to the low affinity TP receptor in human platelets.

Characterization of intestinal gamma-glucoamylase deficiency in CBA/Ca mice.

In previous work, we found that CBA/Ca mice display only 20% of the maltase activity present in other mouse strains. In this study, we characterized more fully the maltase deficiency in CBA/Ca mice. Virtually all of the intestinal maltase activity of CBA/Ca mice was inactivated at 50 degrees C, indicating that it was due only to the sucrase-isomaltase complex. High-performance liquid chromatographic analysis revealed that CBA/Ca mice had undetectable maltase activity displaying the molecular mass characteristic of murine gamma-glucoamylase (gamma-GA) (530 kDa). Gel electrophoretic analysis confirmed that CBA/Ca mice lacked maltase activity with molecular mass of 530 kDa corresponding to gamma-GA. Two-dimensional electrophoretic analysis revealed that the gamma-GA deficiency in CBA/Ca mice was due to the failure to synthesize the enzyme and not to the synthesis of an inactive protein. gamma-GA maltase activity could not be induced in CBA/Ca mice by a diet rich in starch, whereas the activity of other disaccharidases were readily increased. gamma-GA-deficient CBA/Ca mice appear to lack any gross metabolic abnormality resulting from this defect.