Identification and pharmacological characterization of the prostaglandin FP receptor and FP receptor variant complexes.

BACKGROUND AND PURPOSE: A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes. EXPERIMENTAL APPROACH: FP receptor mRNA variants were identified by reverse transcription-polymerase chain reaction. The FP-altFP4 heterodimers were established in HEK293/EBNA cells co-expressing FP and altFP4 receptor variants. A fluorometric imaging plate reader was used to study Ca2+ mobilization. Upregulation of cysteine-rich angiogenic protein 61 (Cyr61) mRNA was measured by Northern blot analysis, and phosphorylation of myosin light chain (MLC) by western analysis. KEY RESULTS: Six splicing variants of FP receptor mRNA were identified in human ocular tissues. Immunoprecipitation confirmed that the FP receptor is dimerized with altFP4 receptors in HEK293/EBNA cells co-expressing FP and altFP4 receptors. In the studies of the kinetic profile for Ca2+ mobilization, prostaglandin F2alpha (PGF2alpha) elicited a rapid increase in intracellular Ca2+ followed by a steady state phase. In contrast, bimatoprost elicited an immediate increase in intracellular Ca2+ followed by a second phase. The prostamide antagonist, AGN211335, selectively and dose-dependently inhibited the bimatoprost-initiated second phase of Ca2+ mobilization, Cyr61 mRNA upregulation and MLC phosphorylation, but did not block the action of PGF2alpha. CONCLUSION AND IMPLICATIONS: Bimatoprost lacks effects on the FP receptor but may interact with the FP-altFP receptor heterodimer to induce alterations in second messenger signalling. Hence, FP-altFP complexes may represent the underlying basis of bimatoprost pharmacology.

Calcium ionophore-induced de-encryption of tissue factor in monocytes is associated with extensive cell death.

INTRODUCTION: Cell surface tissue factor (TF) is normally encrypted, but can be activated by various cellular perturbations. Exposure of TF bearing cells to calcium ionophore has been reported to increase TF activity, de-encrypt TF, by phosphatidylserine (PS)-dependent and -independent mechanisms. Our aim has been to examine at the single cell level, if increased cell surface PS coincided with increased cell surface TF antigen, and cell death (necrosis, 7-AAD-intercalation), and relate this to monocyte- and microparticle (MP)-associated procoagulant activity. MATERIALS AND METHODS: We exposed lipopolysaccharide-stimulated, human, elutriation-purified, cryopreserved TF bearing monocytes to increasing concentrations of calcium ionophore (A23187) and measured procoagulant activity in cells and supernatants. These measurements were compared with quantification of cell surface TF and PS (Annexin V) and of cell necrosis (7-AAD) by flow cytometry, and complemented by confocal microscopy. RESULTS: We observed that calcium ionophore increased cellular and MP-associated TF activity, but not cell surface TF antigen. The discrepancy between TF activity and TF antigen coincided with a dose-dependent increase in the number of cells expressing PS. These cells were to a large extent necrotic and many of them also expressed TF. CONCLUSIONS: We suggest such TF positive dying cells to contribute to the discordance between TF activity and TF expression. Calcium ionophore also increased MP-associated TF activity and release of MPs may be a way to disseminate procoagulant activity. Our findings emphasize the importance of adequately assessing cell death and taking into consideration its possible role in experiments with calcium ionophore.

Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris.

BACKGROUND AND PURPOSE: The prostamides (prostaglandin-ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX-2 from the respective endocannabinoids anandamide and 2-arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists. EXPERIMENTAL APPROACH: The isolated feline iris was used as a key preparation, where prostanoid FP receptors and prostamide activity co-exist. Activity at human recombinant FP and other prostanoid receptors was determined using stable transfectants. KEY RESULTS: In the feline iris, AGN 204396 produced a rightward shift of the dose-response curves for prostamide F2alpha and the prostamide F2alpha analog bimatoprost but did not block the effects of PGF2alpha and synthetic FP receptor agonists. Studies on human recombinant prostanoid receptors confirmed that AGN 204396 did not behave as a prostanoid FP receptor antagonist. AGN 204396 exhibited no antagonism at DP and EP1-4, but was a highly effective TP receptor antagonist. Contrary to expectation, the FP receptor antagonist AL-8810 efficaciously contracted the cat iris. AGN 204396 did not affect AL-8810 induced contractions, demonstrating that AL-8810 and AGN 204396 are pharmacologically distinct. Unlike AL-8810, the ethylamide derivate of AL-8810 was not an agonist. Al-8810 did not block prostamide F2alpha activity. Finally, AGN 204396 did not block PGE2-glyceryl ester activity. CONCLUSIONS AND IMPLICATIONS: The ability of AGN 204396 to selectively block prostamide responses suggests the existence of prostamide sensitive receptors as entities distinct from receptors recognizing PGF2alpha and PGE2-glyceryl ester.

Hypoxia-induced irreversible S-phase arrest involves down-regulation of cyclin A.

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRB) is either functional (T-47D cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). All cells in S phase are immediately arrested upon exposure to extreme hypoxia. During an 18-h extreme hypoxia regime, the cyclin A protein level is down-regulated in cells of both types when in S-phase, and, as we have previously shown, pRB re-binds in the nuclei of all T-47D cells (Amellem et al. 1996). Hence, pRB is not necessary for the down-regulation of cyclin A during hypoxia. However, our findings indicate that re-oxygenation cannot release pRB from its nuclear binding following this prolonged exposure. The result is permanent S-phase arrest even after re-oxygenation, and this is correlated with a complete and permanent down-regulation of cyclin A in the pRB functional T-47D cells. In contrast, both cell cycle arrest and cyclin A down-regulation in S phase are reversed upon re-oxygenation in non-pRB-functional NHIK 3025 cells after prolonged exposure to extreme hypoxia. Our results indicate that pRB is involved in permanent S-phase arrest and down-regulation of cyclin A after extreme hypoxia.

Progesterone concentrations in milk fat at first insemination--effects on non-return and repeat-breeding.

The relationship between milk fat progesterone concentration at first artificial insemination (AI) and reproductive performance of Norwegian Red Cattle dairy cows was investigated in a field study. Fifteen AI technicians collected milk samples from 2250 cows from 458 herds and progesterone was analysed in the milk fat portion of the samples. Logistic regression revealed decreased probability of non-return, and increased likelihood of repeat-breeding, with increasing progesterone concentrations at insemination. The odds ratio for non-return and for repeat-breeding between the minimum and maximum value for milk fat concentrations between 4 and 32.2ng/ml was 4.5 (P < 0.01) and 3.2 (P = 0.01), respectively. The variable 'Technician' did not significantly influence non-return rate or repeat-breeding. Progesterone concentration at AI was positively related to calving to last insemination interval, and to number of inseminations per cow (P < 0.05). The association between the progesterone concentration at insemination and calving interval was only marginally significant. Technician was significantly (P < 0.01) associated with interval from calving to first AI, interval from calving to last AI (P = 0.05), and number of AI per cow (P = 0.01). Technician was not significantly related to the calving interval. We conclude that increased suprabasal progesterone concentrations at the time of first insemination, and higher return rate at AI may, at least partly, be due to endocrinological asynchrony at AI, a condition which may lead to decreased fertility.

Level and distribution of progesterone in bovine milk in relation to storage in the mammary gland.

The study investigated the effect of the place of storage of milk in the mammary gland on progesterone concentrations in whole milk, skim milk and milk fat. Skim milk, milk fat and whole milk progesterone concentrations were lower (P < 0.05) in milk fractions obtained from the cisternal part of the mammary gland compared to those in the milk fractions from the alveoli. Mean milk fat concentrations did not mirror the changes in the mean skim milk, milk fat and whole milk progesterone concentrations. After administration of oxytocin, milk fat concentrations rose significantly (P < 0.01). At the same time, skim milk and milk fat progesterone concentrations remained unchanged (P > 0.05), compared to those in the milk fractions of alveolar origin, obtained before oxytocin administration. Skim milk and whole milk progesterone concentrations were higher (P < 0.01) in composite milk and in milk samples collected 1 h after milking, compared to concentrations in the milk samples collected before morning milking and at 3, 5, 7 and 9 h after milking. The results suggest that defatted milk, milk fat and whole milk progesterone concentrations were affected by the place of storage of the milk in the mammary gland, and that this effect is independent of milk fat content. Time of milk sampling, not the milk fat concentration, in relation to time of milking, was a critical factor in determining skim milk, milk fat and whole milk progesterone. The study also revealed that the concentrations of the other milk components, somatic cell count, lactose and protein were affected by the place of storage of milk in the mammary gland.

Effects of photointensity and photoperiod on milk yield and reproductive performance of Norwegian red cattle.

Relationships between light exposure, milk yield, and reproductive traits in Norwegian Red cattle were studied using data from 1538 farms in southern Norway during the darkest part of the year (November 1 to February 28). Production level was expressed as milk yield at first artificial insemination (AI) and 305-d milk yield. Reproductive performance was calculated using the following dependent variables: age at first AI, age at first calving, days open, calving interval, number of AI per cow, and nonreturn rate at 60 d. Days open and calving interval were both 4.0 d shorter, number of AI per cow was 0.07 less, non-return rate was 3.1% greater, and daily milk yield at first AI was 0.5 kg greater in herds that used dim illumination at night compared with herds that used no illumination at night. Milk yield at first AI was 0.5 kg greater in herds with photoperiods > 12 h. Photoperiods > 12 h were associated with a reduction in age at first AI by 4.8 d and age at first calving by 6.6 d. We concluded that dairy cattle kept at high latitudes during winter should be exposed to dim illumination at night and a minimum photoperiod of 12 h to stimulate reproductive performance and milk yield.