Adenosine triphosphate and adenosine triphosphatase in hormone-containing granules of posterior pituitary gland.

Neurosecretory granules prepared from bovine posterior pituitary glands by cell fractionation methods contain adenosine triphosphate and adenosine triphosphatase activity. Addition of adenosine triphosphate to suspensions of granules stimulates release of vasopressin. It is suggested that adenosine triphosphate and adenosine triphosphatase participate in the storage and release of vasopressin.

Stimulation of adrenal medullary cells in vivo and in vitro induces expression of c-fos proto-oncogene.

The nuclear proto-oncogene, c-fos, has been implicated in the coordinated regulation of gene expression during cell proliferation and differentiation. In this study, we have demonstrated the induction of the c-fos gene products in differentiated cells of the adrenal medulla by non-mitogenic signals. Activation of adrenal medullary cells in vivo by insulin-induced hypoglycemia, and in vitro by nicotine or angiotensin resulted in the rapid and transient elevation of c-fos mRNA levels. Induction of the c-fos mRNA by angiotensin and nicotine were accompanied by the appearance of the c-fos protein. The increase in c-fos protein occurred initially in the cytoplasm and, later, in the nucleus, and it was co-localized with tyrosine hydroxylase. Nuclear expression of the c-fos protein was also induced by veratridine, forskolin and the calcium ionophore A231287. The role of calcium in the regulation of the c-fos gene by angiotensin with nifedipine and inhibition of the effects of angiotensin with nifedipine and sphingosine, a protein kinase C inhibitor. Activation of the c-fos gene may play a role in the coordinated induction of genes involved in the long-term adaptation of adrenal medullary cells to increased functional demands.

Evidence for a role for adenosine 3',5'-monophosphate in progesterone secretion by human chorion.

Experiments were performed to determine whether cells from human chorion can synthesize and release progesterone. Cells were isolated from term chorion laeve by collagenase-DNAse digestion and incubated in RPMI-1640 medium. Freshly isolated cells contained 9.9 +/- 1.1 ng progesterone/10(6) cells, and released 72.0 +/- 7.1 ng/10(6) cells X 24 h in the absence of precursors. When 25-hydroxycholesterol (25HC) served as a precursor, progesterone release into the medium was concentration and time dependent from 1-20 micrograms/ml up to 8 h. When pregnenolone served as a precursor, progesterone secretion followed Michaelis-Menten kinetics (Km = 6.7 microM; maximum velocity, 1.02 nmol/10(6) cells X h). In the presence of 25HC (20 micrograms/ml), progesterone release increased significantly on exposure to cholera toxin (1 microgram/ml), methylisobutylxanthine (0.1 mM), forskolin (0.1 mM), or (Bu)2cAMP (1 mM). Cells maintained in culture released progesterone when fetal calf serum (10%) or 25HC served as precursors. These studies show that trophoblasts from fetal membranes can synthesize and release progesterone from endogenous and exogenous precurors and support the suggestion that cAMP is an important mediator in this process.

Localization of renin in trophoblasts in human chorion laeve at term pregnancy.

It is known that renin is present in fetal membranes, with the highest concentration in the chorion laeve (reflected chorion). The purpose of this study was to identify and localize renin in human chorion laeve. Indirect immunofluorescent analysis, using antiserum against pure human kidney renin, revealed a single layer of cells in the chorion with strongly positive fluorescence. The presence of atrophic villi in this layer together with other morphological evidence indicate that the cells which are positive for renin are cytotrophoblasts. Isolated cells were prepared from the chorion by collagenase digestion, followed by filtration and density gradient centrifugation on Percoll. The isolated cells also showed a positive reaction with the immunofluorescent technique. Control experiments with nonimmune serum did not show fluorescent cells. Biochemical analysis using RIA of angiotensin I generated from sheep substrate indicated that most of the renin activity in the isolated cells was present as inactive renin (activated by trypsin). The presence of renin in trophoblastic cells may be of significance in local cardiovascular regulation, events associated with parturition, or pathophysiological manifestations of trophoblastic disease.

Tumor necrosis factor-alpha and interleukin-1 beta inhibit the synthesis and release of renin from human decidual cells.

Cytokines modulate hormone expression in many cell types, including the expression of renin in juxtaglomerular cells. However, the effect of cytokines on the expression of renin from extrarenal cells is unknown. In this paper, we have examined whether tumor necrosis factor-alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) modulate the release of renin from human decidual cells. Continuous exposure of primary decidual cell cultures from term pregnancies to TNF alpha and IL-1 beta caused dose-dependent inhibition of renin release. The maximal inhibitions by TNF alpha and IL-1 beta were 75.5% and 55.2%, respectively, and the half-maximal effective doses of TNF alpha and IL-1 beta were 30 and 1.1 pmol/L, respectively. The decrease in renin release by the cytokines was statistically significant on days 2-5 (P > 0.001 at each time) and was accompanied by inhibition of renin synthesis and renin messenger ribonucleic acid levels. The renin messenger ribonucleic acid levels in cells exposed for 4 days to TNF alpha (50 ng/mL) or IL-1 beta (50 pg/mL) were 58.0% and 37.7% less than those in control cells, respectively. As decidual macrophages express TNF alpha and IL-1 beta, the results of this study strongly suggest a paracrine role for cytokines in the regulation of decidual renin expression. The effect of these cytokines on renin expression in decidual cells is opposite that in juxtaglomerular cells.

Human chorionic gonadotropin stimulates placental prorenin secretion: evidence for autocrine/paracrine regulation.

Functional regulation of the placental renin-angiotensin system remains incompletely defined. Evidence indicates that synthesis and secretion of prorenin in the placenta and gestational sac decrease with advancing gestational age. Possible explanations for this developmental effect include the regulatory influences by locally released hormones, such as CG. To address this question, the effect of CG on prorenin secretion was evaluated in a human placental explant model. In this study, prorenin concentrations were measured in the media and tissue of cultured explants from term placentas. In addition, the role of cAMP in mediating hormone-regulated prorenin secretion was evaluated. Media and tissue concentrations of prorenin increased (2- and 3-fold, respectively) in a concentration-dependent fashion after 48 h of incubation with CG (0.03-300 IU/ml). Selective inhibition of phosphodiesterases by Ro 20-1724 (type IV) and cilostamide (type III) resulted in a marked potentiation of CG-induced prorenin secretion. Media concentrations of cAMP were also elevated with CG treatment and correlated with prorenin values. Prorenin secretion induced by CG was markedly attenuated by the cAMP-dependent protein kinase inhibitor, H-89. These results indicate that placental prorenin secretion may be locally regulated by CG and mediated by cAMP signal transduction mechanisms.

Beta-adrenoceptor activation stimulates, and phosphodiesterase inhibition potentiates, placental prorenin synthesis and release.

This study evaluated activation of beta-adrenoceptors and the cAMP pathway on prorenin secretion from human placental explants. For comparative purposes, hCG secretion was also measured. Treatment with selective beta-adrenergic agonists (beta 1-dobutamine and beta 2-terbutaline) produced dose-dependent increases in prorenin secretion, with dobutamine yielding a greater response (10- vs. 6-fold). In contrast, hCG secretion was stimulated only by terbutaline (5-fold). Prorenin and hCG secretory responses were inhibited by corresponding selective receptor antagonists (beta 1-metoprolol and beta 2-ICI 118,551). Selective phosphodiesterase inhibitors were used to evaluate the role of cAMP in mediating these responses. Marked potentiation of beta-adrenoceptor-dependent prorenin secretion was observed with the type III inhibitor, cilostamide (63-76%), and the type IV inhibitor, Ro-201724 (32-43%). Type I (8-methoxymethyl-3-isobutylmethylxanthine) and type V inhibitors (dipyridamole and M&B 22,948) showed no potentiation. These studies demonstrate that activation of both beta 1- and beta 2-receptors stimulates placental prorenin release. The potentiation of beta-adrenergically activated prorenin release by selective inhibitors of phosphodiesterase indicates a coupling of beta-adrenoceptor and adenylate cyclase. The contrast in secretion of prorenin and hCG by selective beta-adrenergic agonists suggests differences in cellular localization. The results indicate that clinically used adrenergic agonists can affect the placental renin-angiotensin system. The role of endogenous activators of beta-adrenoceptors in this system remains to be determined.

Human uterine decidual macrophages express renin.

Human uterine decidual tissue contains many cell types, including stromal cells, fibroblasts, and macrophages. Earlier studies have shown that decidualized uterine stromal cells express renin, primarily in the form of prorenin. However, the possibility that decidual macrophages, which comprise about 30% of the cells in term decidua, also express renin has not been investigated. To determine whether macrophages express renin, macrophages were isolated from enzymatically dispersed term decidual cells using immunomagnetic beads coupled to antibodies to human leukocyte antigen (HLA)-DR, an antigen present on macrophages, but not other decidual cells. The isolated cells were 92.1% CD14 positive and contained the messenger ribonucleic acids (mRNA) for the interleukin-2 type alpha receptor, but not for PRL, a specific marker of decidualized stromal cells. Immunocytochemical studies of the macrophage-enriched fraction demonstrated that the macrophages contained renin, and reverse transcription-polymerase chain reaction analysis with primers specific for renin indicated that the fraction also contained renin mRNA. Renin was detected in the conditioned medium of cultures of the macrophage-enriched preparations, greater than 90% of which was in the form of prorenin. As anticipated, renin and renin mRNA were also detected in the HLA-DR negative cells, more than 80% of which stained with specific antiserum to PRL. Peripheral mononuclear cells also expressed renin mRNA, as determined by reverse transcription-polymerase chain reaction analysis. These results demonstrate that human decidual macrophages express renin and indicate that renin is expressed by several cell types in decidual tissue.

Relaxin stimulates the synthesis and release of prorenin from human decidual cells: evidence for autocrine/paracrine regulation.

Porcine relaxin caused a time- and concentration-dependent increase in the release of renin from decidual cells cultured over a 96 h period. The increase in renin release occurred 24-48 h after exposure and was maximal at 48-72 h. Half-maximal stimulation occurred at a relaxin concentration of 5 ng/ml, and maximal stimulation (250-270%) occurred at concentrations greater than or equal to 10 ng/ml. At each time, greater than 95% of the renin released into the medium was in the form of prorenin. The stimulation of renin release was paralleled by a stimulation of cellular renin content and was completely inhibited by cycloheximide, indicating that relaxin also stimulated renin synthesis. Since renin is present in both cytotrophoblast and decidual cells, these results suggest a paracrine and/or autocrine relationship between relaxin- and prorenin-secreting cells.

Stimulation of placental prorenin secretion by selective inhibition of cyclic nucleotide phosphodiesterases.

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 (Ca(2+)/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.

Beta-adrenergic regulation of renin expression in differentiated U-937 monocytic cells.

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 from villous placenta: regulation by cyclic AMP and angiotensin.

Renin synthesis and secretion from human chorion and decidua have previously been shown to be stimulated by agents which increase cellular cyclic adenosine monophosphate (cAMP). We have now used organ culture of villous placenta, incubated for periods up to 72 h, to investigate the cellular regulation of renin in this tissue. The placental tissues release renin (92-96% in the form of prorenin) and human chorionic gonadotrophin (hCG), but not prolactin. We found that cholera toxin and forskolin markedly stimulate the synthesis and release of renin in a time-dependent manner. This stimulation was potentiated by phosphodiesterase inhibitors and inhibited by an angiotensin II agonist, sar-1-angiotensin II. The inhibitory action of the angiotensin agonist on renin release was blocked by sar-1-leu-8-angiotensin II, a selective angiotensin receptor antagonist. The potential for stimulation of renin expression by cyclic AMP-regulated elements is supported by the dramatic (two-orders of magnitude) increase in renin release observed with cholera and forskolin at 72 h. There are several possible candidates for primary signals for adenylyl cyclase-coupled renin secretion from the placenta, including relaxin and epinephrine. The extremely low concentration of renin in term villous placenta may be related to activation of negative regulatory elements on the renin gene. We propose that angiotensin II is one negative regulator of this system.

Cyclic AMP as a second messenger for prorenin release from human decidual cells.

The possible roles of cyclic AMP and protein kinase C in the release of renin from human decidual cells were investigated by examining renin release from monolayers of decidual cells exposed for 72 h to agents that increase intracellular cAMP or activate protein kinase C. Dibutyryl cAMP (10-1000 microM caused a dose-dependent stimulation of renin release after a 24-h exposure. Maximal stimulation, 410 per cent greater than that of control cells, occurred at 72 h, and 98 per cent of the renin released into the medium was in the form of prorenin. Forskolin (10-1000 microM) and cholera toxin (CT. 20-1000 ng/ml), both of which stimulate adenyl cyclase, also stimulated prorenin release. Phorbol myristate acetate (PMA), an activator of protein kinase C, had little effect on basal prorenin release at 100 nM but potentiated the stimulation of prorenin release by cAMP and CT. The effects on prorenin release were paralleled by stimulation of active renin release. The results of this study therefore implicate cAMP and protein kinase C in the regulation of prorenin release from decidual cells and suggest that prorenin release from the decidua and other tissues is regulated by the same second messengers.

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.

Induction of metallothionein in a human trophoblast cell line by cadmium and zinc.

The induction of metallothionein (MT) in a cell line derived from a malignant trophoblastic tumor (JAr cells) was demonstrated using the Cd/heme radioassay following exposure to Cd or Zn. Cd at an optimal concentration of 1 microM produced a 30-fold increase in MT following a 24 hr incubation. Induction by Cd was both time and dose dependent, with a significant increase in MT noted as early as 3 hr, with levels continuing to increase up to 24 hr. Zn was also quite effective in inducing MT synthesis in this cell line. Exposure to 80 microM Zn for 24 hr produced a 70-fold increase in MT. Although Cd was a more potent inducer of MT, exposure to Zn resulted in a greater magnitude of induction. The magnitude of MT induction in JAr cells was much greater than that seen in cultured trophoblasts from term chorion laeve. The degree of induction seen in this cell line makes it an interesting model for the study of MT's role in trophoblast function. MT induction in trophoblasts may reflect a protective mechanism against heavy metal toxicity and/or an integral aspect of normal zinc homeostasis.

Regulation of utero-placental prorenin.

Prorenin (Pro) is synthesized in a number of human utero-placental tissues, including chorion, decidua, villous placenta and probably mesenchymal cells. The release of Pro from these extra-renal tissues follows new protein synthesis and appears to utilize the constitutive secretory pathway. Unlike processing in the kidney, very little of the Pro is subsequently cleaved to the smaller product (active renin). Primary signals which regulate Pro include protein hormones and peptides (relaxin, endothelin, hCG), amines (epinephrine, norepinephrine, and related beta adrenergic agents), and eicosanoids. These agents increase the mRNA for prorenin at a time before peak secretory effects are noted. Other extracellular signals have negative regulatory effects. These include angiotensin, endotoxin and cytokines (TNF-alpha and interleukin-1 B). There is also evidence that glucocorticoid receptor activation has an inhibitory effects on Pro release in placenta. Second messengers involved in the regulation of Pro include cyclic AMP and protein kinase A (PKA), protein kinase C (PKC), and calcium. The possible biological effect(s) of the extracellular Pro are unknown but may be due to direct generation of angiotensin I. Since angiotensin-peptides have a number of trophic effect on both vascular and non-vascular tissues, regulation of utero-placental Pro by autocrine, paracrine or endocrine signalling may be critical in normal fetal and/or placental development.