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Fat embolism (FE) and the consequent FE syndrome occurring after trauma or surgery can lead to serious pulmonary injury, including ARDS and death. Current treatment of FE syndrome is limited to supportive therapy. We have shown in a rat model that the renin angiotensin system plays a significant role in the pathophysiology of FE because drugs interfering with the renin angiotensin system, captopril and losartan reduce the histopathologic pulmonary damage. The purpose of the current study was to determine if inhibition of renin by aliskiren, an FDA-approved drug for treating hypertension, would produce effective protection in the same model.The FE model used intravenous injection of the neutral fat triolein in unanesthetized rats. Intraperitoneal injections of saline or aliskiren at either 50 or 100 mg/kg were performed 1 hour after FE induction via triolein. Rats were euthanized at 48 hours, and various histologic stains were used to examine the lungs.(1) Fibrosis: rats treated with triolein showed significant fibrotic changes with increased collagen and myofibroblast activation (p0.0001 for both trichrome and α-smooth muscle actin staining). Aliskiren blocked this inflammatory and profibrotic process to a level indistinguishable from the controls (p0.0001 for both trichrome and α-smooth muscle actin staining). (2) Fat: rats treated with triolein showed a statistically significant increase in fat (p = 0.0006). Subsequent aliskiren administration at both doses reduced the size, distribution, and amount of fat droplets (low dose, p = 0.0095; high dose, p = 0.0028). (3) Vessel patency: the low dose of aliskiren blocked the reduction of lumen patency observed after triolein administration (p = 0.0058).Aliskiren protected the lungs of rats from gross and histopathologic FE-induced pulmonary damage at 48 hours. Clinical implications include the use of aliskiren both prophylactically (before certain orthopedic procedures) and therapeutically (after severe trauma) to prevent the consequent severe pulmonary pathologic sequelae.

Background Fat embolism (FE) after trauma and some orthopedic procedures is known to cause acute lung injury, including acute respiratory distress syndrome. However, its potential long-term effects on the lung are unknown. A previous study using a rat model of FE found significant histopathologic changes in the lungs after intravenous injection of triolein for up to 11 days. This study detailed the persistence of the lung damage and investigated the input of the renin-angiotensin system in its pathology. Methods Unanesthetized rats were injected via the tail vein with 0.2 mL saline or triolein. After euthanasia, at 3 weeks or 6 weeks, lung sections were stained to highlight cellular structure, presence of collagen and fat, or immunolabeled for smooth muscle actin or angiotensin peptides. Results At 3 weeks or 6 weeks after triolein injection, there was no dilatation of the heart or inferior vena cava, no congestion of the liver or spleen, no adventitial edema, nor was fluid present in alveoli or pleural cavity as reported in animals at earlier time points. Persisting pathology included reduced lumen patency, thickening of the media of small arteries and arterioles, and vascular and septal inflammation. Although the fat content of the lung decreased from week 3 to week 6, there was a progressive increase in collagen, smooth muscle actin, and angiotensin peptides. Conclusions This model extends the effect of FE on pulmonary pathology to 6 weeks, revealing persistent vasculitis, septal inflammation, and progressive fibrotic changes which are associated with increased presence of angiotensin peptides.

Fat embolism syndrome (FES) is a lung process following bone fracture, surgery, and liposuction, expressed by inflammation, vasculitis, and fibrosis. A rat model of FES is induced by IV injection o...

We present the case of a 29 year-old primigravid obese female with a complex medical history, who underwent caesarean section at 35 weeks gestation due to preeclampsia. She has a past medical history of bilateral foot surgery, and bilateral hip pinning for chronic hip pain in 2012. Postoperatively, approximately six hours after surgery, she was in respiratory distress and 47 hours later she was found dead. At autopsy, a diagnosis of pulmonary fat embolism was made. Pulmonary histopathological examination revealed findings similar to an animal model of fat embolism.

BACKGROUND: FE, predominantly a respiratory disease, is thought to be the result of fat globules entering the bloodstream from tissue that has been disrupted by trauma but scarce information about ...

In a model of triolein-induced fat embolism (FE) in the rat, we have reported that the acute pulmonary histopathological changes were ameliorated by losartan (Los). The chronic pulmonary changes of FE persisted at 6 weeks and symptoms were exacerbated by a “second hit” at that time using LPS. The present experiment extended FE study to 10 weeks and examined whether the FE effects plus LPS would continue to be blocked by Los. Groups of rats were given FE (triolein i.v. or i.v. saline controls) followed 6 weeks later by LPS (3 mg/kg i.p). One-hour after LPS rats were treated with saline or Los (2 mg/kg i.p. and then 0.1 mg/ml Los in the drinking water for 4 more weeks). In confirmation of our previous results, the FE group showed normal weight gain both at 6 and 10 weeks and appeared to be in no distress. However the lungs showed reduced lumen patency at 10 weeks, accompanied by inflammatory and fibrotic changes that were greater than what had been found previously at 6 weeks whether or not LPS was added. T...

Previously our laboratory has shown that 17β-estradiol in vivo rapidly decreases R(+)-8-OH-DPAT-stimulated [35S]GTPγS binding (a measure of the initial biochemical event in the intracellular signaling pathway associated with 5-HT1A receptors) in the hippocampus, frontal cortex and amygdala. Studies were designed to determine if 17β-estradiol also acts in vitro on estrogen receptors in the hippocampus and frontal cortex to decrease 5-HT1A receptor function. Hippocampus and frontal cortex were dissected from ovariectomized rats and incubated for up to 3 h with various estrogens and antiestrogens; membrane homogenates were prepared for R(+)-8-OH-DPAT-stimulated [35S]GTPγS binding assays. 17β-Estradiol (10–6 M) decreased the maximal response in the R(+)-8-OH-DPAT-stimulated [35S]GTPγS binding assay in a time-dependent manner (observed at 30, 60 and 120 min) in both hippocampus and frontal cortex. The hormone, however, did not alter the EC50 of R(+)-8-OH-DPAT. When hippocampus and frontal cortex were incubated in graded concentrations of 17β-estradiol for 1 h, the calculated EC50 was approximately 2.5 × 10–8 M in both brain regions. The nonestradiol estrogen diethylstilbestrol also decreased 5-HT1A receptor function while the less potent estrogens 17α-estradiol and estriol were inactive at 5 × 10–8 M. The estrogen receptor antagonist ICI 182,780 potently and completely blocked the effects of 17β-estradiol on 5-HT1A receptor function with an apparent KB of approximately 10–9 M. These data demonstrate clearly that estrogens can act on estrogen receptors located in hippocampus and frontal cortex of ovariectomized rats to produce rapid heterologous decreases in 5-HT1A receptor function.

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.

Histological lung damage in the model of fat embolism (i.v. triolein) is ameliorated by agents acting on the renin-angiotensin system (RAS), with pulmonary damage persisting up to 6 wks. Here we re...

Experiments were performed to examine the effect of interferon-y (IFNy) on the expression of renin by human uterine decidual cells and decidual macrophages. Exposure of a mixed population of decidual cells consisting of 80% decidualized stromal cells and 20% macrophages to IFNy for 4 days caused a dose-dependent inhibition of renin release beginning 2 days after exposure. Renin release on Day 4 was inhibited by a maximum of 83.9%, and the half-maximal effective dose of IFNy was 5 ng/ml (290 pM). The inhibition of renin release in response to IFNy was accompanied by a comparable inhibition of renin mRNA levels. In addition to inhibiting basal renin expression, IFNy potentiated the inhibitory effect of tumor necrosis factor a (TNFa) on renin expression. IFNy also inhibited basal renin release and potentiated the inhibitory effect of TNFa by highly purified populations of decidual stromal cells and decidual macrophages prepared by immunomagnetic separation with beads coupled to an anti-human leukocyte antigen (HLA-DR) antibody that binds macrophages but not stromal cells. Reverse transcription-polymerase chain reaction analysis showed that HLA-DR(+) cells express IFNy mRNA, and that both HLA-DR( +) and HLA-DR( -) cells express IFNy receptors. Since IFNy is expressed only by decidual macrophages, the results of this study strongly suggest that IFNy inhibits the expression of decidual renin by a paracrine action.

The pathophysiology of Fat Embolism Syndrome (FES) is poorly understood and subject to some controversy. Evaluation of the evolution of histological changes in the lungs of patients with FES is impractical. The current theories of FES were established through acute clinical observations and acute animal experiments, but sequential changes in the histology of lungs over a prolonged period have not been made. The progressive effects of fat embolization of the lungs were examined in a rat model over a period of 11 days. Triolein, a major bone marrow fat, was administered to conscious Sprague-Dawley rats via the caudal vein. Rats were euthanized at 24, 48, 96 h, and 11 days, but some died within a few hours. Histomorphometric evaluations of lung tissue were made, including stains for fat, collagen, and smooth muscle actin. Arterial and arteriolar patency decreased progressively up to 96 h, but returned toward normal after 11 days. A striking finding was the very early presence of inflammation and fibrosis after only several hours, persisting up to 11 days. The results of this study provide evidence of both very early and prolonged changes due to fat embolization.

The human placenta and related tissues are considered to be examples of the recently accepted local renin–angiotensin systems (RAS). The brain is another example of a system that is thought to be regulated independently of the kidney and the role of angiotensin within the CNS as a neural mediator has drawn considerable attention. It has been known for a long time that many of the neuroendocrine mediators and receptors are expressed in the placenta and it has been suggested that there are many parallels between the classical neuroendocrine system and the placental one. The present review summarizes information that components of the RAS are expressed in uteroplacental tissues, are regulated by endogenous substances, and have important biological functions within this reproductive system. A comparison of similarities and differences between the classical and the placental RAS may provide clues to functions in other endocrine and neuroendocrine systems. The major components of the placental RAS that are considered are renin, prorenin, angiotensin I, angiotensin II, angiotensin converting enzyme (ACE), angiotensin receptors, and angiotensinogen (renin substrate). The factors that regulate these components at the cellular and the nuclear level are described. It is concluded that prorenin via angiotensin-dependent and angiotensin-independent mechanisms influences functions within uteroplacental tissues. Some of these actions are direct and others are mediated by the release of different signalling molecules. These features are similar to many neuroendocrine systems and utilize some of the same messengers.

Activation of beta-adrenoceptors has been shown to promote renin secretion in both human kidney and placenta. In kidney, the enhanced secretion is immediately observed, and mobilization of renin in the storage granules accounts for such a rapid response. In contrast, the enhanced secretion in placenta is delayed for 6-12 hr after receptor activation and consists almost entirely of the renin precursor prorenin. It is hypothesized that newly synthesized rather than stored enzyme is responsible for the enhanced secretion in human placenta. To test this hypothesis, placental explants were cultured in the presence or absence of the protein synthesis inhibitor cycloheximide, and prorenin concentrations in the tissue and medium were measured. Dobutamine and terbutaline, beta1- and beta2-adrenoceptor agonists, evoked 17- and 5-fold increases in secretion, respectively. Tissue content of prorenin in response to the treatment was increased by a similar magnitude, yet values were consistently

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.

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.

OBJECTIVE: Term villous placental concentrations of prorenin are known to be very low, whereas those of decidua and fetal membranes are high. It has been demonstrated that prorenin synthesis is modulated by hormones in othe reproductive tissues, thus suggesting a means for paracrine regulation in the placenta. This study was performed to test the hypothesis that placental tissue prorenin concentrations may be influenced by gestational age and are temporally related to alterations in the hormonal milieu. STUDY DESIGN: Decidua and villous placental tissue were obtained from term and first-trimester human pregnancies, and concentrations of prorenin, active renin, prolactin, and human chorionic gonadotropin were measured. Values were compared between gestational periods, and relationships between renin and hormone values were analyzed. RESULTS: Prorenin concentrations in first-trimester placenta were nearly 200-fold higher than at term. The proportion of active renin was higher with early gestation. Decidual prorenin and active renin concentrations were similar in both groups. Placental prorenin correlated with chorionic gonadotropin but not prolactin in both groups. CONCLUSIONS: This study demonstrates large differences in placenta prorenin and active renin in villous placental tissue between first-trimester and term gestation tissues, yet there differences were not observed in decidual tissues. The contrast in placental prorenin values observed at the extremes of pregnancy parallel those of placental human chorionic gonadotropin.

Summary 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.

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.

The purpose of this study was to examine the effects of angiotensin on the release of enkephalin peptides and the expression of the proenkephalin (pEK) gene. Incubation of cultured bovine adrenal medullary (AM) cells in serum-free medium resulted in calcium- and time-dependent accumulation of [Met 5 ]-enkephalin (MEK) in the medium. Fifteen minutes to three hours of incubation with 2 n M [Sar 1 ]-angiotensin II (s 1 -AII) did not affect basal secretion of MEK; however, longer incubations (24 h) resulted in four- to fivefold increases. Northern and dot blot analyses with bovine pEK cDNA demonstrated increases in the relative abundance of pEK mRNA in angiotensin-treated cells, suggesting that the long-term increases in MEK release may reflect increased expression of pEK gene and MEK synthesis. Stimulation of MEK release and induction of pEK mRNA were concentration dependent (ED 50 approximately 1 n M . Changes in pEK mRNA levels were not observed until 12 h of incubation with s 1 -AII and continued to increase during an additional 12 h of incubation. Addition of an angiotensin antagonist, saralasin, at 0–16 h, but not at 18–20 h, to cells incubated continuously for 24 h with s 1 -AII inhibited changes in pEK mRNA and MEK release. These observations demonstrate the absence of apparent desensitization of angiotensin receptor function and indicate that long-term receptor-ligand interactions are required to induce changes in gene expression and MEK release. Induction of pEK mRNA and stimulation of MEK release were additive to the effects of veratridine and forskolin, respectively, indicating that the effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Angiotensin-induced increases in pEK mRNA were partially inhibited by nifedipine and also by dantrolene and TMB-8, drugs that inhibit voltage-dependent calcium channels and mobilization of calcium from intracellular stores, respectively. s 1 -AII-induced changes in pEK mRNA were inhibited with calmidazolium, suggesting involvement of calmodulin. The participation of protein kinase C in the induction of pEK gene and long-term stimulation of MEK release was indicated by inhibition of the s 1 -AII effects by pretreatment of cells with protein kinase C inhibitor sphingosine. Effects of s 1 -AII on induction of pEK mRNA by angiotensin and by nicotine were prevented by the translational inhibitor cycloheximide. In conclusion, angiotensin receptors were found to control expression of the pEK gene and secretion of MEK. Unlike nicotinic receptors, which may control secretion of enkephalin peptides directly by stimulating exocytosis and indirectly by controlling peptide synthesis, the effects of angiotensin appear to be mediated indirectly at the level of pEK gene expression.

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.

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.

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.

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.

SummaryDithiothreitol (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.

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.

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.