F. Russo-Marie

Characterization and partial purification of ‘renocortins’: two polypeptides formed in renal cells causing the anti‐phospholipase‐like action of glucocorticoids

1 Anti‐inflammatory steroids reduce prostaglandin E2 (PGE2) synthesis in rat renomedullary interstitial cells in culture by inhibiting the release of arachidonic acid from membranous phospholipid stores, exhibiting antiphospholipase‐like properties. 2 After treatment of the cells with dexamethasone 10−6M, these cells release a protein in the supernatant. 3 This supernatant is able to inhibit PGE2 secretion in untreated cells and to inhibit phospholipase A2 activity in an in vitro system. 4 Using chromatofocusing separation, we showed that two distinct proteins exist with isoelectric points of 5.8 and 8.3. 5 Using gel permeation separation, we showed that two proteins exist with apparent molecular weights of 15,000 and 30,000 daltons. 6 We conclude that, in renal cells in culture, anti‐inflammatory steroids induce the synthesis and the release of two polypeptides which we have named ‘Renocortins’ (induced by corticoids in renal cells) causing the antiphospholipase‐like action of glucocorticoids. 7 Our results are in good agreement with others, but as renal cells are not directly involved in the inflammatory process, we suggest that this steroid‐induced phenomenon is not solely involved in the inflammatory reaction but is of more general physiological relevance.

Dexamethasone-induced inhibition of prostaglandin production does not result from a direct action on phospholipase activities but is mediated through a steroid-inducible factor

Investigations were carried out to define the mechanisms of steroid-induced inhibition of prostaglandin secretion by rat renomedullary cells in tissue culture. Although it was strongly proposed that glucocorticoids may inhibit phospholipase A2 activity, we present several pieces of evidence against a direct action of dexamethasone on phospholipase activities. First, dexamethasone, which significantly decreases the release of labeled material from cells prelabeled with [3H]arachidonate, does not significantly alter the pattern of distribution of the radioactivity among the various classes of cell lipids. In addition, direct measurement of phospholipase A3 activity in dexamethasone-treated cells failed to show any significant decrease in the deacylation capacity. On the other hand, several indications suggest that dexamethasone may induce the secretion of a non-dialysable, transferable factor able to inhibit prostaglandin production, the mechanism of which remains to be investigated.

Differential of sex steroids of prostaglandin secretion by male and female cultured piglet endothelial cells

Abstract The effect of sex steroids, 17β-estradiol and testosterone, on the production of 6-keto-prostaglandin F 1α , prostaglandin F 2α and prostaglandin E 2 was studied in cultures of piglet aorta endothelial cells. In cells isolated from female animals both steroids stimulated the secretion of prostaglandins. In contrast, sex steroids did not affect prostaglandin synthesis by endothelial cells taken from male animals. In addition, female endothelial cells convert testosterone into Estriol, estrone and estradiol. estradiol-induced stimulation of prostacyclin production may explain in part the beneficial role generally attributed to naturally occuring estrogens in cardiovascular diseases.

6-Ketoprostaglandin F1α, prostaglandins E2, F2α and thromboxane B2 production by endothelial cells, smooth muscle cells and fibroblasts cultured from piglet aorta

After [3H]arachidonic acid labeling, cyclooxygenase products were qualitatively analysed in the media of each cultured vascular cell type by reverse-phase high-performance liquid chromatography (rp-HPLC). The prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha and thromboxane B2 detected in the rp-HPLC radioactive profile were then quantified by radioimmunoassay (RIA) in separate sets of experiments. In preconfluent endothelial cells prostaglandin F2 alpha and 6-ketoprostaglandin F1 alpha were detected in equal amounts (49%), whereas after confluence 6-ketoprostaglandin F1 alpha represented 57% of total secretion (P less than 0.05). Smooth muscle cells secreted mainly prostaglandin F2 alpha (48%) and fibroblasts prostaglandin E2 (44%). Using the bioassay method, antiaggregatory activity was detected only in endothelial cells, though a small percentage of immunoreactive 6-ketoprostaglandin F1 alpha was encountered in smooth muscle cells and fibroblasts (13 and 10%, respectively). Radioimmunological analysis after rp-HPLC separation of the medium of endothelial cells showed that the anti-6-ketoprostaglandin F1 alpha antibody recognized, among other substances, an unidentified compound. Its retention time was similar to that of prostaglandin F2 alpha. This unidentified compound was not detected in the media from smooth muscle cells and fibroblasts.

Prostanoid synthesis by vascular slices and cultured vascular cells of piglet aorta

Abstract Biosynthesis of prostanoids was studies in vascular slices of human umbilical arteries, piglet aorta and vena cava as well as in cultured vascular cells of piglet aorta. After preincubation with radioactive labeled arachidonic acid, prostanoids in the incubation media of slices or cultured cells were measured by radioimmunoassay or by radioactivity determination of labeled compounds following separation on reserved-phase high performance liquid chromatography. In all vascular slices 6-keto-PGF1α was the main prostanoid found, followed by PGE2. Thromboxane B2 and PGF2α were also formed, but only in trace amounts. In cultured cells taken from the three layers of the vascular wall, the prostanoid profiles differed markedly from those obtained from vascular slices. Each cell strain showed a specific prostanoid pattern. Endothelial cells synthesized predominantly 6-keto-PGF1α and PGF2α. In smooth muscle cells no 6-keto-PGF1α could be detected; here the predominant prostanoid was PGE2. PGF2α was formed in smaller quantities. Fibroblasts synthesized all prostanoids (PGE2, PGF2α, TXB2, 6-keto-PGF1α), PGE2 and PGF2α being the major products. In vascular slices and in cultured endothelial cells, the predominant prostacyclin derivative detected was 6-keto-PGF1α; the enzymatic PGI2-metabolite, 6,15-diketo-PGF1α, could be detected only in piglet vena cava slices in small amounts.

Effect of dexamethasone on cyclo-oxygenase activity in reno-medullary cells in culture

The steroid-induced inhibition of prostaglandin secretion has been shown to be mediated through the interaction of the steroid with specific receptors and to require RNA and protein synthesis. At present the nature of the protein(s) involved is unknown although several suggestions have been made for a role of phospholipase A2. It has also been postulated that the early action of steroid is a stimulation of cyclo-oxygenase activity leading only secondarily to an inhibition of arachidonate supply. In this paper, we have therefore investigated the effect of dexamethasone on cyclo-oxygenase activity in cultures of rat reno-medullary interstitial cells. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone induces a moderate increase in cyclo-oxygenase activity but this elevation does not appear sufficient to account for the inhibitory action of dexamethasone on PG secretion.

Angiotensin II does not elicit any specific prostaglandin secretion in piglet cultured endothelial cells

PGE2 and PGF2 alpha were measured in culture media from piglet aortic endothelial cells by radioimmunological analysis. Prostacyclin secretion was evaluated by radioimmunological analysis of its stable metabolite, 6-keto-PGF1 alpha after reverse-phase high pressure liquid chromatography separation. No stimulation of either prostaglandin was detectable in culture media after treatment with angiotensin II (10(-9) to 10(-6) M) for 15 to 120 min at 37 degrees C. Under the same conditions angiotensin II (10(-7) M) elicited a 2 to 3 fold increase in PGE2 and PGF2 alpha secretion when incubated with cultured piglet aortic smooth muscle cells. In addition, we failed to detect specific angiotensin receptors at the surface of intact cultured endothelial cells. Since there was a very rapid increase in prostaglandin secretion after washing or medium changes we suggested that the effects of Angiotensin II on prostacyclin production, demonstrated in perfused organs, could be due to the mechanical stimulation elicited by the contraction of the underlying smooth muscle cells.

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture

Cultured vascular smooth muscle cells from porcine aortas incubated in Na+-free medium rapidly release their intracellular Na+ contents (Nai) (23±4% of baseline after 60 min incubation, mean ± SEM of 18 experiments). Total Nai release was inhibited by 35–40% after addition of ouabain and by 60–70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na+ efflux with an IC50 of about 10−9–10−8 M. Addition of the alpha-adrenergic agonist phenylephrine (10 μM) to the cells mimicked the inhibitory action of norepinephrine on Nai release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Nai release. Simultaneous addition of 10 μM norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Nai decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 μM) inhibited 40.0±8.1% of ouabain-sensitive Rb+ influx and 70.7±6.9% of bumetanide-sensitive Rb+ influx (mean ± SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb+ influx was obtained with about 5×10−7 M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na+, K+, Cl−]-cotransport system is able to catalyze outward Na+ movements (in Na+-free media) of a similar order of magnitude to those of the Na+, K+ pump and that alpha-adrenergic stimulation markedly inhibits Na+ efflux (and Rb+ influx) through these two transport systems.

Mechanism of Steroid-Induced Inhibition of Prostaglandin Production by Rat Renomedullary Cells in Culture

The mechanism by which steroids induce the inhibition of prostaglandin biosynthesis is now rather well documented. If it is generally accepted that steroids at high doses are able to stabilize cell membranes,1,2 this stabilizing property cannot account for the steroid’s ability to interfere with prostaglandin synthesis. Several authors reported that steroids inhibit the synthesis of prostaglandins by decreasing the release of arachidonic acid from the membranous phospholipids and consequently its availability for cyclooxygenase.3–6 We7 and others8–11 have shown that the anti-inflammatory effects of steroids are mediated through receptor occupancy7 and require RNA and protein synthesis.7–11 The nature and the identification of the protein(s) involved are now under study: Blackwell et al.12 and Hirata et al.13 have described the synthesis of polypeptides induced by anti-inflammatory steroids. One of these polypeptides has been described in rat macrophages by Blackwell et al.12 The polypeptide which was named “macrocortin” has a molecular weight of 15,000 and is able to inhibit phospholipase A2 activity in intact cells. Hirata et al.13 described another polypeptide, “lipomodulin,” synthetized by rabbit polymorphonuclear neutrophils, whose molecular weight is 45,000. The debate is still continuing as to whether these peptides are similar, i.e., whether lipomodulin is the precursor of macrocortin.