Upper limits on perturbations of nuclear decay rates induced by reactor electron antineutrinos.

We report the results of an experiment conducted near the High Flux Isotope Reactor of Oak Ridge National Laboratory, designed to address the question of whether a flux of reactor-generated electron antineutrinos (ν¯e) can alter the rates of weak nuclear interaction induced decays of 54Mn, 22Na, and 60Co. This experiment has small statistical errors but, when systematic uncertainties are included, has null results. Perturbations greater than one part in 104 are excluded at 95% confidence level in ß± decay and electron capture processes, in the presence of an antineutrino flux of 3 × 1012 cm-2s-1. The present experimental methods are applicable to a wide range of radionuclides. Improved sensitivity in future experiments can be anticipated as we continue to better understand and reduce the dominant systematic uncertainties.

Particulate ANP-sensitive guanylyl cyclase in blood and bone marrow cells of patients with acute leukemia.

Increases in plasma cyclic GMP levels have been shown to correlate with increased plasma levels of atrial natriuretic peptide (ANP) in patients with fluid overload due to increased secretion of ANP. There is evidence that plasma cyclic GMP levels are also elevated in some patients with acute leukemia, but increased ANP secretion has not been demonstrated. To elucidate the possibility that a newly expressed guanylyl cyclase may be responsible for the increase of plasma cyclic GMP levels patients with acute and chronic leukemia as well as patients with lymphoma and healthy volunteers were studied. Plasma levels of cyclic GMP were measured and isolated peripheral blood mononuclear or bone marrow cells were incubated with increasing concentrations of ANP. The stimulation of cells was measured as cGMP accumulation in the supernatant. Furthermore guanylyl cyclase activity was measured in membrane preparations of peripheral blood mononuclear cells. While leukocytes of healthy subjects were devoid of detectable ANP-stimulated particulate guanylyl cyclase activity, ANP-sensitivity was observed in seven patients with acute lymphoblastic and in three patients with acute myelogenous leukemia. Cyclic GMP in the supernatant of cells was elevated between 2- and 132-fold of basal when cells were incubated with 1 microM ANP for 60 minutes. Like in healthy volunteers, no effect of ANP on freshly isolated mononuclear cells was observed in cases with chronic leukemia or in patients with lymphoma. Expression of ANP-sensitive particulate gunaylyl cyclase may be connected with malignant transformation of lymphocytes in patients with acute leukemia and might be useful for their diagnosis and classification.

Particulate ANP-sensitive guanylyl cyclase: induction in cultured human peripheral CD3+ mononuclear blood cells.

There have been conflicting reports about the occurrence and/or activity of atrial natriuretic peptide (ANP) sensitive guanylyl cyclase in the immune system. This study reports on ANP-sensitive guanylyl cyclase mRNA expression and guanylyl cyclase activity in human peripheral blood mononuclear cells (PBMC). Reverse transcription polymerase chain reaction (RT-PCR) shows that activated human PBMC of healthy blood donors express functional active ANP-sensitive guanylyl cyclase after vitro culture, whereas freshly isolated PBMC show neither specific mRNA for particulate guanylyl cyclase nor ANP-sensitive activity of this enzyme. To define the subpopulation of PBMC expressing this enzyme, cultivated PBMC were subfractioned and analyzed by RT-PCR and in situ PCR. Only CD3+ PBMC showed mRNA for ANP-sensitive guanylyl cyclase. Induction of the guanylyl cyclase required coincubation with other cells, indicating that a factor or factors secreted from cells other than CD3+ cells induces this expression. In summary, ANP-sensitive guanylyl cyclase is an inducible enzyme in human CD3+ PBMC in contrast to other cells where it is considered to be constitutive.

Effect of glycosylation on cloned ANF-sensitive guanylyl cyclase.

Cloned ANF-sensitive guanylyl cyclase (GC-A) and ANF-sensitive guanylyl cyclase from adrenal cortex differ in their sensitivity to the ANF analogs atriopeptin 1 and urodilatin. To test the hypothesis that these differences are due to different glycosylation, we investigated the effect of the N-glycosylation inhibitor tunicamycin on GC-A. Tunicamycin altered the response of GC-A to atriopeptin 1 and urodilatin, whereas the sensitivity to ANF remained unchanged. These data indicate that agonist specificities of different ANF-sensitive guanylyl cyclases are influenced by carbohydrate moieties.

Atrial-natriuretic-peptide receptors in glomerular cryosections of renal malignant and spontaneously hypertensive rats.

ANP-receptors affinities (KD) and capacities (Bmax) were assayed in cryosections of glomeruli from 'malignant' hypertensive rats (2K-1C) and spontaneously hypertensive rats (PHR). Plasma ANP concentration was twofold higher in 2K-1C (P < 0.05) and PHR (P < 0.02) than in the respective controls, KD and Bmax for rANP99-126 and ANP103-123 did not differ. ANP mediated cGAMP release in 2K-1C rats was also unaffected. ANP-C glomerular receptors (i.e. displacement of tracer binding with ANP103-123) were not down-regulated and had unchanged peptide binding affinity in either kidney of rats with 'malignant' hypertension and in PHR. The difference between Bmax for rANP99-126 and Bmax for rANP103-123 (ANP-A receptor binding) indicates moderate up-regulation of ANP-A receptors in the clipped, and down-regulation in the contralateral kidney of 2K-1C (2K-1C, right vs. left, P < 0.05). Since [ANP]pl, and also Bmax and KD for ANP were similar in both hypertension models investigated, changes of the [ANP]pl/ANP-receptor system can not completely explain the marked natriuresis of rats with 'malignant' hypertension.

A non-isotopic immunoassay for guanosine 3':5'-cyclic monophosphate using a cyclic GMP-biotin conjugate as tracer.

2'-O-monosuccinylguanosine 3':5'-cyclic monophosphate was coupled to N-biotinyl-1,8-diamino-3,6-dioxaoctane after converting succinyl-cGMP into its N-hydroxysuccinimide active ester. Isolation and purification of the succinyl-cGMP-biotin conjugate was performed with FPLC using reversed phase chromatography. The synthesis described yielded a conjugate suitable for use as tracer in immunoassays for the cGMP measurement in plasma and urine samples. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for flexible end point determination by means of commercially available labeled streptavidin derivatives. Streptavidin-europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA), streptavidin-horseradish peroxidase was used for colorimetric end point determination (EIA). Both non radioactive immunoassay systems showed excellent correlation with the reference radioimmunoassay, good sensitivity and reproducibility. The succinyl-cGMP-biotin conjugate was shown to be stable for more than two years without any apparent loss of chemical stability or immunological reactivity.

Distribution of membrane bound guanylyl cyclases in human intestine.

The quantification and distinction of particulate guanylyl cyclases in the human intestine were considered by an enzymatic approach, which comprised the signal transduction from receptor binding to cGMP formation, and, in addition, by showing the expression of an intracellular portion of these transmembrane proteins. Basal guanylyl cyclase (GC) activities were 50 to 80 pmol cGMP formation/min/mg protein and were stimulated up to twofold by heat stable enterotoxin, but were not significantly influenced by atrial natriuretic factor. Enzymatic analysis of colonoscopic specimens pointed to the prevalence of guanylyl cyclase C in the terminal ileum and in the large bowel including colon ascendens, colon descendens, sigmoid, and rectum. The availability of sequence information on human guanylyl cyclases permitted the development of a polymerase chain reaction approach for distinguishing the expression of GC-A and GC-C in human tissue samples. The expression levels of particulate guanylyl cyclases found by polymerase chain reaction in surgical biopsy specimens confirmed the enzymatic data, in that substantial expression of GC-C was found not only in the small intestine but also in the large bowel. According to the restriction mapping of amplificates, GC-C prevailed over GC-A throughout the human intestine, particularly in the mucosal layers.

Plasma cGMP level as a marker of the hydration state in renal replacement therapy.

We investigated, whether plasma cyclic guanosine 3':5'-monophosphate (cGMP) may be suited as a marker of hyperhydration in hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD). In 81 HD patients the levels of atrial natriuretic peptide (ANP) and cGMP were markedly elevated before HD (ANP: 165 +/- 11.1 pg/ml; cGMP 43.5 +/- 2.2 pmol/ml). Significantly lower values (P < 0.01) were found after HD (ANP: 97 +/- 8.4 pmol/ml; cGMP 19.5 +/- 1.5 pmol/ml). Twenty-three patients had cGMP levels above 20 pmol/ml after HD. Therefore "dry body weight" was reduced in these patients. This resulted in a "normalization" of cGMP values to a postdialytic range below 20 pmol/ml in the majority of patients. All seven patients with persistently high cGMP levels despite weight reduction had left sided heart failure. In 33 CAPD patients ANP was slightly lower than after HD (68 +/- 10.4 pg/ml), and the cGMP level (22.4 +/- 2.3 pmol/ml) was between pre- and postdialytic values in HD. In eight CAPD patients with clinical signs of hypervolemia plasma cGMP, but not ANP, was significantly elevated. We conclude that the plasma cGMP level appears to be a reliable marker for fluid overload in patients on renal replacement therapy with normal heart function.

The circulating bioactive form of human guanylin is a high molecular weight peptide (10.3 kDa).

Guanylin is a peptide isolated from rat intestine that stimulates intestinal guanylate cyclase. We describe here the purification of circulating guanylin from human hemofiltrate. By N-terminal protein sequence analysis 47 amino acids were determined. This sequence corresponds to the positions 22 to 68 of the prohormone deduced from the cDNA sequence of human proguanylin. Mass spectral analysis of the circulating peptide showed the molecular weight to be 10,336 Da, which corresponds to the mass calculated from position 22 to the C-terminus of the peptide predicted from the cDNA sequence. Circulating guanylin markedly increased the cyclic GMP content of T84 cells. Our data show that the hormonal form of guanylin is circulating as a 10.3-kDa peptide in human blood.

Comparison of a cloned ANF-sensitive guanylate cyclase (GC-A) with particulate guanylate cyclase from adrenal cortex.

Recently, an ANF-sensitive guanylate cyclase (GC-A) has been cloned from a rat brain cDNA library. Here we studied the stimulation of cyclic GMP accumulation in response to atrial natriuretic factor (ANF), urodilatin and atriopeptin I (AP-1) in a rat glioma C6 cell line permanently transfected with GC-A as well as GC-A activity in membranes from these C6 cells and in membranes from COS-7 cells that were transiently transfected with GC-A. We also measured binding affinities for these natriuretic peptides in the membrane preparations. These characteristics of GC-A were compared to those of membrane preparations from adrenal cortex of bovine and human origin. The order of potency of stimulation of cyclic GMP accumulation in permanently transfected glioma cells was ANF greater than urodilatin greater than AP I; AP I stimulated cyclic GMP accumulation. A similar order of potency was obtained for stimulation of guanylate cyclase activity in membranes from permanently transfected glioma cells as well as from transiently transfected COS-7 cells. In contrast, AP-1 was uneffective to stimulate guanylate cyclase in membrane preparations from adrenal cortex from bovine as well as from human origin. Furthermore, urodilatin was equipotent to ANF in these preparations. Binding affinities were comparable for ANF and urodilatin in membranes from cells transfected with GC-A and in membranes from adrenal cortex of both sources, whereas AP-1 had a weaker affinity in all preparations studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and expression of a guanylate cyclase-coupled heat stable enterotoxin receptor cDNA from a human colonic cell line.

Heat stable enterotoxins (STs) are low molecular-weight peptides secreted by enterotoxigenic bacteria. One type of these enterotoxins (STa) induces intestinal secretion leading to acute diarrhea by binding to a membrane form of guanylate cyclase. We have isolated a cDNA from a human colonic cell line, T84, encoding for a guanylate cyclase-coupled enterotoxin receptor (STaR). The predicted amino acid sequence of the human STa receptor is 81% identical with the previously cloned enterotoxin receptor (GC-C) from rat intestine. COS-7 cells transiently transfected with the cloned cDNA expressed specific concentration-dependent response to STa as measured by cyclic GMP accumulation and is about 20 times more sensitive to the stimulation by STa than has been shown for GC-C.

Comparison of anti-platelet properties of molsidomine, isosorbide-5-mononitrate and placebo in healthy volunteers.

The purpose of the present work was to investigate the ex vivo platelet-inhibiting properties of the nitric oxide-containing vasodilator, molsidomine, and the organic nitrate, isosorbide-5-mononitrate, in comparison with placebo. Ex vivo platelet aggregation in 11 healthy volunteers was measured before, as well as 30 and 60 min after, the intake of either 4 mg molsidomine, 20 mg isosorbide-5-mononitrate (ISMN) or placebo in a randomized double-blind fashion. The release of thromboxane was also determined. Threshold doses of platelet-activating factor (PAF) to induce irreversible aggregation were significantly increased by 100 and 120% 30 and 60 min after molsidomine. Slopes of aggregation curves (aggregation induced with 50 and 200 nM PAF) were significantly reduced after molsidomine (P less than 0.01). Small platelet-inhibiting effects were also observed after ISMN and after placebo intake. The release of thromboxane was not influenced when platelets were maximally stimulated either during clotting of whole blood or during aggregation of platelet-rich plasma with a high dose of PAF. Thromboxane release with a low dose of PAF was reduced 30 and 60 min after drug intake, independent of whether molsidomine, ISMN or placebo was applied. The data indicate that the usual clinical doses of molsidomine, but not of ISMN inhibit platelet aggregation in healthy man.

[Platelet aggregation with SIN 1: comparison with isosorbide-5-mononitrate and acetylsalicylic acid]. / Thrombozytenaggregation unter SIN 1: Vergleich mit Isosorbid-5-mononitrat und Acetylsalizylsäure.

SIN 1, the bioactive metabolite of molsidomine, not only appears to lack the problem of inducing nitrate tolerance, but also exerts antiaggregatory and fibrinolytic properties. These effects, which either are not or only in part shared by the drug isosorbide-5-mononitrate, might be beneficial in the prevention of thromboembolic complications in cardiovascular disease. In contrast to the effects of acetylsalicylic acid, SIN 1 already inhibits aggregation during the first phase of aggregation, and it inhibits aggregations induced by agonists that are not or only marginally influenced by acetylsalicylic acid (such as the aggregation induced by platelet activating factor). Thus, the antiaggregatory effects of molsidomine cannot replace the effects of acetylsalicylic acid, while a combination of both drugs might be of benefit in the treatment of patients with cardiovascular disease.

Functional modulation of ANF-sensitive particulate guanylate cyclase by redox mechanisms.

We examined the effects of sulfhydryl-reactive redox agents and of the apparent oxidation-reduction (redox) potential of the assay medium on enzyme activity and atrial natriuretic factor (ANF) binding properties of particular guanylate cyclase from bovine adrenal cortex. Redox potential was varied by addition of redox-reactive agents and quantified by electrochemical measurement. The modification of free SH groups by thiol-reactive agents had only a minor effect on ANF binding or on the extent of ANF-dependent enzyme stimulation whereas free thiol groups were essential for basal enzyme activity of ANF-sensitive particulate guanylate cyclase. Basal and ANF-stimulated particulate guanylate cyclase activity was modulated by exposure to different redox potential states. This modulation was different for the substrates Mg.GTP and Mn.GTP. The apparent redox potential had no influence on the extent of guanylate cyclase activation by ANF. Our results suggest that critical free thiol groups, which are sensitive to thiol-reactive redox agents, are involved in the catalytic, but not in the receptor function of ANF-sensitive particulate guanylate cyclase. These thiol groups could be the structural basis for the effects of redox events which modulate basal enzyme activity, but not activation by ANF.

Kinetic characterization of atrial natriuretic factor-sensitive particulate guanylate cyclase.

The present investigation describes kinetic characteristics of membrane-bound and Triton X-100-solubilized atrial natriuretic factor (ANF)-sensitive guanylate cyclase from bovine adrenal cortex. The kinetic analysis of both enzyme forms suggests that in the presence of manganese, ANF induces or stabilizes at least two apparent GTP*Mn2(+)- and in addition two Mn2(+)-binding sites. Addition of the natriuretic drug amiloride favors this state. ATP increases the vmax in the presence of ANF for GTP*Mg2+, but not for GTP*Mn2+ as a substrate. With GTP*Mg2+, amiloride has no effect on basal or ANF-stimulated activity, but slightly reduces the effect of ATP. Under all conditions tested, the enzyme follows regular Michaelis-Menten kinetics in the presence of Mg2+ and exhibits positive cooperativity with Mn2+. Positive cooperativity is also retained after Triton extraction. The results indicate that Triton extraction has no major influence on the kinetic properties of particulate guanylate cyclase when the extraction procedure is done carefully. The data also support the suggestion that multiple interactions of subunits might occur upon activation of the enzyme by ANF in the presence of Mn2+.

Effects of a small bolus dose of ANF in healthy volunteers and in patients with volume retaining disorders.

Thirty-seven patients with volume-retaining disorders (liver cirrhosis with ascites, n = 8; heart failure NYHA III-IV, n = 12; endstage renal failure, n = 17) and twelve healthy age-matched controls were given a small dose (33 micrograms) of hANF (human atrial natriuretic factor). We tested the resulting hemodynamic and renal effects as well as the effect on plasma cyclic GMP levels and compared them with the properties of platelet ANF receptors. The ANF injection evoked an increase in cyclic GMP plasma levels of 19.3 +/- 2.2 nM in healthy controls. This increase tended to be smaller in the cirrhosis group (15.5 +/- 3.3 nM) and in the heart failure group (16.8 +/- 2.3 nM) than in the dialysis group (20.5 +/- 2.5 nM). The invasion rates of cyclic GMP were comparable in all groups, but the evasion rates increased more in the heart failure and endstage renal failure groups (27.9 +/- 7.7 min and 26.1 +/- 3.4 min, respectively) than in the cirrhosis and control groups (14.9 +/- 1.9 min and 14.2 +/- 1.9 min, respectively). Patients with endstage renal failure and congestive heart failure showed a smaller decrease in diastolic blood pressure than controls and patients with liver cirrhosis. Renal actions of ANF were diminished in cirrhosis and heart failure patients. Binding capacities of platelet ANF receptors were higher in the control group (12.2 +/- 1.5 receptors/cell) than in the patient groups (cirrhosis, 7.8 +/- 1.2; endstage renal failure, 8.0 +/- 0.9; heart insufficiency, 8.0 +/- 1.0 receptors/cell), with no differences among the patient groups. Binding affinities were not significantly different. Correlation analysis showed that the relationship between the actions of ANF and the increases in plasma cyclic GMP levels is loose and cannot predict the hemodynamic or renal effects of exogenous ANF in a given patient. Although the behavior of plasma cyclic GMP levels fails to predict the responsiveness of the body to ANF in a given patient, it does reflect the differences between the patient groups and the control group. In contrast, we found no correlation between the properties of platelet ANF receptors and ANF action.

[Inhibition of platelet aggregation by endothelium-derived relaxing factor-like agents]. / Hemmung der Thrombozytenaggregation durch EDRF-ähnliche Wirkstoffe.

In the last years, an inhibition of aggregation by organic nitrates or by similar drugs has been demonstrated by some authors, but has also been ruled out by other authors. The present work was thus performed to study a possible inhibition of platelet aggregation by the respective drugs in comparison with the molecular mechanism of action of these drugs, that is activation of soluble guanylate cyclase. We found that in vitro, organic nitrates activate soluble guanylate cyclase and inhibit platelet aggregation only in millimolar concentrations, while sodium nitroprusside and SIN-1, the active metabolite of molsidomine, influence these parameters in micromolar concentrations. This difference between the actions of the O-NO2-containing nitrates and the NO-containing compounds nitroprusside and SIN-1 is, however, not apparent ex vivo. Ex vivo, not only molsidomine, that is converted in the liver to SIN-1, but also isosorbide-5-mononitrate inhibited platelet aggregation. Thus, it appears that organic nitrates can in vivo release nitric oxide in a tissue other than platelets in amounts that are high enough to inhibit platelet aggregation. These studies suggest, that an antiaggregatory effect may participate in the clinical actions not only of drugs that directly resemble EDRF, such as SIN-1, but also by the organic nitrates. However, since nitrates cannot be activated directly by the platelets, it appears that also the antiaggregatory effects of nitrates, but not of molsidomine, underlie the mechanisms of tolerance development.

Effect of molsidomine on ex vivo platelet aggregation and plasma guanosine 3':5'-cyclic monophosphate levels in healthy volunteers.

To find out whether 3-morpholino-sydnonimine (SIN 1), the active metabolite of molsidomine, exerts its antiaggregatory effects not only in vitro but also in vivo, we tested ex vivo aggregation before and after intravenous application of molsidomine in healthy volunteers. We also measured plasma levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) as SIN 1, the bioactive metabolite of molsidomine, becomes effective via activation of soluble guanylate cyclase. In eight out of ten subjects molsidomine had an inhibitory effect on platelet aggregation and a higher threshold concentration of platelet-activating factor was required after molsidomine application to induce irreversible aggregation. Despite the effect on platelets, plasma cyclic GMP levels did not increase. These results suggest that the nitric oxide-containing SIN 1 inhibits platelet aggregation not only in vitro but also in vivo and that this property can be a beneficial effect in antianginal therapy.