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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.

Prorenin secretion by human villous placenta is known to be stimulated by activation of adenylate cyclase and enhanced cyclic AMP (cAMP) generation. Placental tissue contains predominantly type III (cGMP-inhibited) and type IV (cAMP-specific) phosphodiesterases (PDEs), which inactivate cAMP. To evaluate the role of PDE subtypes in the regulation of prorenin secretion by human placenta, explants were cultured in the presence of isobutylmethylxanthine (IBMX), a non-selective PDE inhibitor, and selective inhibitors for various PDE subtypes. Inhibition of PDE subtypes with cilostamide (type III), Ro 20-1724 (type IV) and zardaverine (types III and IV) increased prorenin release. Inhibition of type I (Ca2+/calmodulin-dependent) PDE by 8-MeoM-IBMX and of type V (cGMP-specific) PDE by zaprinast or dipyridamole did not affect prorenin secretion. The stimulation of prorenin secretion by PDE inhibitors was attenuated by cAMP-dependent protein kinase inhibition. The selective PDE inhibitors caused a parallel increase in media cAMP and prorenin and also increased tissue prorenin levels. These studies demonstrate that cAMP degradation by type III and IV PDE isoenzymes is a major regulatory mechanism for placental prorenin secretion. It is suggested that enhancers of adenylate cyclase activity are constitutively present in placenta and influence prorenin synthesis and release.

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.

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.

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.

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.

Bovine adrenal glands, perfused in vitro , responded to single injections or continuous infusions of aminophylline by increased release of catechoiamines. This effect was not mediated by the release of acetylcholine in the gland, since it was not blocked by cholinergic blocking agents. Stimulation for short periods of time (4 min or less) showed that the catecholamine release was depressed when extracellular calcium was reduced; however, even in the absence of calcium, aminophylline still evoked catecholamine release. The stimulant effect of aminophylline was present during perfusion with high potassium media and was inhibited by the local anesthetic, dibucaine. Papaverine potentiated the response to aminophylline. The results are discussed in terms of two different mechanisms for aminophylline-induced secretion, one dependent on influx of extracellular calcium, and one dependent on release of intracellular calcium.