Alan M. Poisner

Release of inactive renin from human fetal membranes and isolated trophoblasts.

Human chorio-amnion from term pregnancy was perfused in vitro in a dual perfusion apparatus. Renin released from the fetal membranes was almost entirely in the form of inactive renin (IR) (trypsin-activated). IR was released from both the chorion side and the amnion side. IR introduced on the chorion side of the chorio-amnion did not appear on the amnion side. When cells isolated from term amnion and chorion were grown in tissue culture, IR was released continuously from chorionic cells but not from amnionic cells. The release of IR did not parallel the release of LDH from cultured chorion cells. Exposure to low calcium medium (with or without EGTA) decreased IR release while LDH release increased (or remained constant). Exposure to the calcium ionophore (A 23187) resulted in a marked decrease in IR release. The release of IR from chorionic cells shows some similarities to the release of HCG from trophoblasts. It is expected that IR release from the chorion will show some similarities to R release from the kidney as well as major differences. The function, regulation, and processing of chorionic IR remain to be elucidated.

Direct stimulation of renin release by calcium.

Summary Calcium directly stimulates renin release from rat kidney slices previously treated with calcium-free medium. The stimulant effect of calcium (0.5 or 6.0 mM) is not seen without the period of calcium depletion. The stimulant effect of calcium is still present in sodium-free medium but is reduced when the incubation is performed at 20° instead of 37°. The results suggest that the underlying mechanism of renin release may be comparable to that of catecholamine release, involving calcium-dependent and energy-dependent steps. We acknowledge the valuable technical assistance of Mr. John Gillespie and Mrs. Roselle Poisner.

Partial purification and chromatographic properties of inactive renin from human amniotic fluid

Abstract Inactive renin from human amniotic fluid was partially purified using gel filtration and several types of affinity chromatography. The Chromatographie steps employed resulted in a 62-fold purification of inactive renin with an overall yield of 11 per cent. Inactive renin was retained on an Affi-Gel Blue column and could be eluted with 1.4 M NaCl. Chromatography on a concanavalin A—agarose affinity column yielded two fractions. The non-retained fraction contained the inactive renin while the retained fraction contained cathepsin D. Acid or pepsin activation of the non-retained inactive renin, followed by concanavalin A chromatography, resulted in the retention of 33 per cent of the applied renin activity. Inactive renin was also not retained on a pepstatin affinity column. However, after the activation of the inactive renin with pepsin, the enzymatic activity was bound to the pepstatin column. Gel filtration on Sephacryl S-200 indicated that inactive renin had a molecular weight of 39,000. No change in molecular weight was observed after activation with pepsin.

Storage and Release of Renin and hCG in Trophoblast from Human Chorion Laeve

We have recently demonstrated by immunocytochemical and biochemical methods that the trophoblast layer of the term chorion laeve and isolated chorionic cells contain renin (Poisner et al., 1981a). We have also shown that cultured chorionic cells continue to synthesize and release renin for periods up to six weeks (Poisner et al., 1982a). Since the principal cell in the trophoblast layer is the cytotrophoblast (Bourne, 1962), we have examined two other potential cell markers of trophoblast, hCG and progesterone. The origin of hCG in the placenta is generally considered to be the syncytiotrophoblast (Friesen, 1973), although cells with the appearance of cytotrophoblast or transitional cell types have also been reported to contain hCG (Thiede and Choate, 1963; Patillo et al., 1971). Progesterone has also been found in trophoblast, but the localization of hCG and progesterone in the chorion laeve has not been well studied.

Inhibition of pseudorenin by pepstatin.

Abstract The pentapeptide pepstatin was found to inhibit the ability of rat spleen pseudorenin to form angiotensin I from tetradecapeptide renin substrate. Dixon and Webb plots showed that this inhibition was noncompetitive in nature. Lineweaver-Burk analysis also showed noncompetitive inhibition. K i values determined by the three graphical methods ranged from 1.8 to 3.8 × 10 −10 M. The K m for pseudorenin was determined to be between 0.82 and 1.23 μM. The concentration of enzyme used was estimated to be 3.1 × 10 −10 M. Pepstatin should prove useful in the future for the analysis and purification of pseudorenin.

Evidence that pseudorenin activity in bovine spleen is due to cathepsin D

Abstract Pseudorenin and cathepsin D activity from bovine spleen were found to behave identically on DEAE-cellulose, Sephadex G-100, and concanavalin A-agarose chromatography. The molecular weight of pseudorenin and of cathepsin D was estimated to be 50,000. The binding of the enzymatic activity to concanavalin A-agarose and elution with α-methyl- d -mannoside indicates that pseudorenin (cathepsin D) is a glycoprotein. It is suggested that pseudorenin activity in the spleen is due to cathepsin D.

Properties of renin granules isolated from rat kidney

Renin granules from rat kidney prepared at 25 degrees C show greater stability at 25 degrees C than at 0 degrees C when incubated in ionic medium. The sum of the renin in the supernatant fluid plus that in the pellet was the same at 25 degrees C as at 0 degrees C, thus ruling out the possibility that the extra release at 0 degrees C merely represented greater stability of free renin at 0 degrees C. In common with other secretory granules, renin granules were most stable at pH 6.0 and were osmotically sensitive. In contrast to neurosecretory and chromaffin granules, renin granules were stabilized by Mg-ATP in ionic medium. This result is similar to studies by others on lysosomes. It is concluded that the renin granules membrane shares many of the properties of other granule membranes. Some of these properties (temperature and pH lability) will have to be considered in the design of future experiments on renin storage and release.

Renin Release from Human Chorionic Trophoblasts in Vitro: The Role of Cyclic Amp and Protein Kinase C

Trophoblasts from human chorion laeve contain renin (Poisner et al., 1981b), HCG (Poisner et al., 1983a) and progesterone (Tonkowicz and Poisner, 1985). Most of the renin in the chorion is present in the form of prorenin (Poisner et al., 1981b). Preliminary work indicated that renin release is dependent on external calcium and can be inhibited by agents which block calcium influx (Poisner et al., 1983b; 1984a,b). This suggested that renin release may be mediated through the common calcium-dependent exocytosis pathway. Progesterone secretion from these cells is enhanced by dibutyryl cyclic AMP and agents which increase cyclic AMP (Tonkowicz and Poisner, 1985). Cyclic AMP has also been suggested as a mediator of renin secretion from the kidney (Fray, 1980), but the role of calcium is controversial (Chen and Poisner, 1976; Park et al., 1981; Hinko et al., 1984). Another mediator of secretion in a number of cells is the diacyl glycerol-activated protein kinase (C-kinase), which is also activated by the tumor promoter phorbol esters (Castagna et al., 1982). The present studies were carried out to determine if cyclic AMP and protein kinase C may also function as mediators of renin secretion from chorionic trophoblasts. Preliminary studies have been reported (Poisner, 1984; Poisner et al., 1984b).

Plasma pseudorenin in rats after alteration in the renin-angiotensin system.

Summary Plasma levels of renin and pseudorenin were measured at various times in control rats, and after bilateral nephrec-tomy, treatment with propranolol, or the converting enzyme inhibitor (CEI) SQ 20881. After nephrectomy or propranolol, renin levels decreased but pseudorenin levels increased. Following treatment with CEI, renin levels rose in a nonoperated animal and remained very low in a nephrecto-mized animal. Pseudorenin levels did not change appreciably after CEI in either animal. We acknowledge the valuable technical assistance of Mr. John Gillespie and Mrs. Roselle Poisner. We wish to thank E. R. Squibb for the generous supply of SQ 20881.

Dithiothreitol Augments Renin Activity by an Action on Renin Substrate

Summary Dithiothreitol (DTT) increases the generation of angiotensin when bovine renin acts on bovine substrate or hog substrate. Kinetic analysis indicates that the Vmax increases without a change in Km. Pretreatment of bovine renin with DTT followed by dilution of the DTT to a low concentration does not augment renin activity, while pretreatment of the substrate and a similar dilution reveals marked potentiation. N-Ethylmaleimide (NEM) does not inhibit renin activity but does prevent the augmenting effect of DTT present during the incubation. It is concluded that DTT augments renin activity by an action on renin substrate and that renin does not fall in the category of sulfhydryl-dependent enzymes. We acknowledge the valuable technical assistance of Mr. Paul Arnold and Mrs. Roselle Poisner.