Study of prostaglandin receptors in mitochondria on apoptosis of human lung carcinoma cell line A549.

PGs (prostaglandins) are synthesized through the cyclo-oxygenase (COX-1 and -2) pathway in a variety of cells in response to various physiological stimuli. All cells require at least one pathway for apoptosis, and mitochondrial play a central role in regulation of apoptosis. In a previous study, incubation of A549 cells with NS-398 (a COX-2-specific inhibitor) induced apoptosis and inhibited cell proliferation, and the concentrations of different PGs between various cellular compartments were found to be changed. To determine whether PG receptors are involved in this regulation, Western-blot analyses were performed specific for PGE(2) (EP receptors) and PGF(2alpha) (FP receptor) receptors, which were expressed in A549 cells. Western-blot analysis revealed that mitochondria that were isolated from A549 cells expressed EP receptors (EP2, EP3 and EP4), whereas FP receptors were undetectable. EP receptors (EP1, EP3 and EP4) and FP receptors were detected from A549 cell membrane. These results suggest that the change of PG production in A549-cells-induced cancer cell apoptosis might be related to the different expressions of EP and FP receptors in cell and mitochondrial membrane.

Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney.

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.

Monoclonal antibodies that inhibit the enzyme activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

Three hybridoma cell lines secreting antibodies against human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) were produced. Purified IgG2b from these cell lines recognized a distinct band of Mr 28,000 on SDS/PAGE from the purified enzyme as well as a band of Mr 56,000 from the crude enzyme preparation. These three monoclonal antibodies inhibited 15-OH-PGDH activity to different degrees. Inhibition of the enzyme activity could be prevented by prior incubation of the enzyme with NAD+ but not with prostaglandin E2 (PGE2) or NADP+. Inhibition by monoclonal antibodies appears to be non-competitive with respect to NAD+ and PGE2. An increased concentration of antibodies alters the apparent Km for NAD+ but not for PGE2, further supporting the notion that the antibodies bind to the coenzyme-binding site. The availability of these monoclonal antibodies should be valuable for probing the structure of the active site.

Prostacyclin production in vascular endothelium of patients with Blackfoot disease.

The levels of 6-keto-PGF1 alpha and TXB2 were determined by the RIA method in Blackfoot disease which is an endemic disease of the peripheral vascular system found in the southwest coast of Taiwan. The level of TXB2 was normal, but that of 6-keto-PGF2 alpha concentration was 30% lower than the normal. The activity of 15-hydroxyprostaglandin dehydrogenase was higher in patients' blood plasma than in the normal. Patient's vascular endothelium was also found to have lower prostacyclin synthase activity. These results suggest that the reduced prostacyclin production in vascular endothelium contributes to the pathogenesis of Blackfoot disease.

Monoclonal antibody against the catalytic subunit of bovine heart cAMP-dependent protein kinase and the application of enzyme purification.

A monoclonal antibody against the catalytic subunit of bovine heart cAMP-dependent protein kinase has been prepared. The antibody, M73/PK1, belongs to the heavy-chain subclass IgG3 and has a titer of 20,480. Purified M73/PK1 was obtained by ammonium sulfate precipitation and Protein A-agarose affinity chromatography and was coupled to CNBr-activated Sepharose 6MB. The catalytic subunit was purified by this affinity column. A 75% yield and 4746-fold purification were obtained. These results show that this is a suitable and effective method for the purification of the catalytic subunit of bovine heart cAMP-dependent protein kinase.

Effects of arsenate and ergot alkaloid compounds on prostacyclin synthesis in human umbilical endothelium.

Cultured human umbilical endothelium was incubated with various concentrations of o-arsenilic acid and ergotamine tartrate respectively for 72 hr at 37 degrees C, 5% CO2/95% air in 100% humidity. At the end of incubation, medium was removed for the determination of 6-keto-PGF1 alpha concentration by RIA method. Compared to the normal controls, arsenate (0.1 to 10.0uM) showed inhibition (17 to 24%) on the PGI2 production. Ergotamine tartrate gave an activatory effect (20 to 12%) in low concentration (0.1 to 1.0uM) incubation, but had inhibitory effect (25%) on the PGI2 production in higher concentration incubation (10.0uM). These results indicate arsenate in low and high concentrations does not play an important role on prostacyclin synthesis in human umbilical endothelium. However, ergotamine tartrate at higher concentration might be a main factor for the lower prostacyclin synthesis in Blackfoot disease, an endemic disease of the peripheral vascular system found in the southwest coast of Taiwan.

The primary prostaglandin-inactivating enzyme of human placenta is a dimeric short-chain dehydrogenase.

The native form of NAD-dependent 15-hydroxyprostaglandin dehydrogenase of human placenta has a mol. wt. of about 50 000, while the subunit mol. wt. is around 28 000, suggesting a dimeric quaternary structure. These properties, the amino acid composition, insensitivity to EDTA, and inhibition patterns show general similarities to other short-chain dehydrogenases. Several hormones tested did not influence the activity of 15-hydroxyprostaglandin dehydrogenase, but an unusual activation by two anti-depressant drugs was found and may relate to the existence of a natural regulatory factor.