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Previous studies from our laboratories demonstrated that human decidual macrophages and peripheral mononuclear cells express renin. In the present study, we found that U-937 monocytes, induced to differentiate into macrophage-like cells by treatment with phorbol dibutyrate (PDBU), express renin mRNA and release renin (95%, of which is in the form of prorenin). Treatment of these PDBU-exposed cells with dibutyryl-cAMP (1 mM) caused a 20-fold increase in renin mRNA and a 10-fold increase in prorenin release. Forskolin (10 microM), an activator of adenylyl cyclase, and terbutaline (100 microM), a beta2-adrenergic agonist known to increase cAMP levels, also increased renin mRNA and prorenin release. The secretory response to terbutaline was potentiated by the type IV cyclic AMP-phosphodiesterase (PDE) inhibitor Ro 20-1724 (50 microM). Angiotensin II agonist inhibited the stimulatory effect of terbutaline on renin secretion as did the cytokines tumor necrosis factor-alpha and lipopolysaccharide plus interferon-gamma. Since other studies have shown that U-937 cells possess beta2-adrenergic receptors and express mainly the type IV PDE, the present findings strongly suggest that beta-adrenergic receptors in mononuclear cells are coupled to renin expression via the cAMP transduction pathway. The results support a possible role for the renin-angiotensin system in macrophage function and suggest potential autocrine regulatory mechanisms in prorenin expression.

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.

The Chromatographic and kinetic properties of acid- and pepsin-activated inactive renin from human amniotic fluid were determined. Acid-activated inactive renin, like inactive renin, was found to be bound to an Affi-Gel Blue affinity column. Pepsin-activated inactive renin, on the other hand, did not bind to such a column. The K m values of endogenous active renin, acid-activated inactive renin, and pepsin-activated inactive renin with bovine substrate were 0.11, 0.12 and 0.05 μM respectively. With hog substrate, the K m values were 0.16,0.19 and 0.10 μM respectively. These results suggest that there is a difference between the active form of renin obtained from acid activation of inactive renin and the active renin obtained after pepsin treatment.

Metallothionein (MT) levels were measured in the term human placenta, chorion, and amnion by the Cd-radioassay method. MT concentrations in the amnion and chorion were 8 to 9 μg MT/g tissue while placental levels were approximately half this value. For individual subjects a significant correlation ( r 2 = 0.96) was found for placental and chorionic MT, while amnionic MT levels did not correlate with either placental or chorionic MT. Gel filtration and uv spectral analysis confirmed the presence of a MT-like protein in cytosol obtained from amnion, chorion, and placenta. MT levels in trophoblasts cultured from chorion were increased upon exposure in vitro to either Cd or Zn.

Bovine adrenal glands were perfused in vitro or in situ with Locke's solution and were stimulated with acetylcholine (ACh). Catecholamine efflux rose sharply in response to acetylcholine but there was no change in the efflux of phospholipids or cholesterol, substances that are known to be present in the membranes of the chromaffin granules. The results support the view that the membranes of the chromaffin granules remain in the cell during catecholamine extrusion.

Publisher Summary This chapter elaborates a study analyzing mechanisms of exocytosis. Exocytosis is a method of discharge of secretory products in which there is selective release of granule-bound material. Other forms of secretion may cause nonselective release of cellular constituents. In the adrenal medulla, for instance, elevated pH causes the release not only of catecholamines but also of a cytoplasmic marker, lactic dehydrogenase (LDH). This is probably indicative of cell damage. Reserpine can release catecholamines without discharge of other soluble granule constituents. This represents another mechanism of catecholamine release and is also different from exocytosis. Evidence that exocytosis occurs in the adrenal medulla in response to the physiological transmitter, acetylcholine, and other drugs have been reviewed. This chapter discusses some of the factors believed to be involved in exocytisis in the adrenal medulla. The chapter highlights that inhibitors of ATP synthesis increase the spontaneous release of catecholamines from the adrenal gland, while inhibiting the evoked release. These findings have been interpreted along with the evidence that ATP activates calcium uptake by cell organelles and that ATP can release catecholamines from isolated chromaffin granules.

Summary The presence of tubulin in the adrenal medulla is demonstrated by colchicine binding, vinblastine (VB) precipitation, electrophoresis and antigenic activity. Tubulin in the supernatant fraction of the adrenal medulla is similar to that in rat brain supernatant: it has the same electrophoretic mobility and is equally activein complement fixation by the antisera to bovine brain tubulin. Some differences are found: VB precipitation of tubulin is less selective and less complete with the adrenal medulla supernatant than with rat brain; also, there is smaller amount of colchicine binding by the particulate fraction from adrenal medulla than with rat brain. It is proposed that tubulin has similar functions in the adrenal medulla and brain; and further that it is involved in the mechanisms of release.

Purified fractions of chromaffin granules from the adrenal medullae of cats have been prepared for an electron microscopic study of changes associated with the release of catecholamines. Fractions from unstimulated medullae consisted of electron-opaque, membrane-bounded spheroidal granules of about 4500 A maximum diameter, similar to those seen in situ . Fractions from animals whose adrenal medullae had been stimulated with acetylcholine and depleted of catecholamines consisted almost entirely of electron-translucent structures. It is concluded that during release of catecholamines from the chromaffin granules, the granules discharge their electron-opaque contents, but not their membranes, which remain in the cell as discrete structures.

Subcellular fractions obtained from bovine adrenal medullae stimulated to secrete catecholamines were compared with corresponding fractions from unstimulated medullae. Stimulation (with acetylcholine) caused a fall in the catecholamines and protein in the ‘crude granule fractionrs, but no change in the phospholipid or cholesterol. Further resolution of the crude granule fraction by density gradient centrifugation revealed a redistribution of the phospholipid and cholesterol after stimulation from the denser to the lighter layers. The results are interpreted to mean that the membranes of the chromaffin granules remain in the chromaffin cells after catecholamine secretion as discrete particles with reduced specific gravity. Probably these particles are the electron-translucent granules observed by electron microscopy in stimulated chromaffin cells.

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.