An ester bond linking a fragment of a serine proteinase to its serpin inhibitor

Most known members of the serpin superfamily are serine proteinase inhibitors. Serpins are therefore important regulators of blood coagulation, complement activation, fibrinolysis, and turnover of extracellular matrix. Serpins form SDS-resistant complexes of 1:1 stoichiometry with their target proteinases by reaction of their P1-P1' peptide bond with the active site of the proteinases. The nature of the interactions responsible for the high stability of the complexes is a controversial issue. We subjected the complex between the serine proteinase urokinase-type plasminogen activator (uPA) and the serpin plasminogen activator inhibitor-1 (PAI-1) to proteolytic digestion under nondenaturing conditions. The complex could be degraded to a fragment containing two disulfide-linked peptides from uPA, one of which included the active site Ser, while PAI-1 was left undegraded. By further proteolytic digestion after denaturation and reduction, it was also possible to degrade the PAI-1 moiety, and we isolated a fragment containing 10 amino acids from uPA, encompassing the active site Ser, and 6 amino acids from PAI-1, including the P1 Arg. Characterization of the fragment gave results fully in agreement with the hypothesis that it contained an ester bond between the hydroxyl group of the active site Ser and the carboxyl group of the P1 Arg. These data for the first time provide direct evidence that serine proteinases are entrapped at an acyl intermediate stage in serine proteinase-serpin complexes.     

Purification and identification of a novel and four known serine proteinase inhibitors secreted by human glioblastoma cells

Our previous studies have shown that some human cancer cell lines produce pancreatic trypsinogen, plasminogen, and tissue-type kallikrein. To understand the regulatory mechanism of these proteinases, serine proteinase inhibitors secreted by human glioblastoma cell line T98G were analyzed by gelatin reverse zymography with trypsin. The serum-free conditioned medium of T98G cells showed more than ten trypsin inhibitor bands ranging from 16 to 150 kDa in the reverse zymography. Major trypsin inhibitors were purified by trypsin-affinity chromatography. Analysis of their N-terminal amino acid sequences demonstrated that the purified inhibitors were identical to the secreted forms of amyloid protein precursors (APPs), tissue factor pathway inhibitor (TFPI), placental protein 5 (PP5)/TFPI-2, and secretory leukocyte proteinase inhibitor (SLPI). In addition, a novel 25-kDa trypsin-binding protein, tentatively named p25TI, was identified. p25TI showed weak inhibitory activity against trypsin in reverse zymography as compared with the other inhibitors. The secretion of multiple forms of serine proteinase inhibitors by human cancer cells raises the possibility that they might be involved in the abnormal growth of cancer cells.     

Prognostic value of plasma atrial natriuretic factor, norepinephrine and epinephrine in acute myocardial infarction

Case reports from the United Kingdom (UK) in 1989 have suggested that the introduction of human insulin in 1985 was associated with an increased risk of sudden death in insulin-treated diabetic patients. If human insulin increases the risk of sudden death, the number of these should have increased during the period where human insulin was introduced. We therefore identified all cases of sudden death in Denmark in younger insulin-treated diabetic patients, age at death below 50 years. During this period the consumption of human insulin went from 0.2% to 70% of the total consumption in Denmark. The total number of cases fulfilling the inclusion criteria was 226, and the annual number of sudden deaths did not change during the study period (p = 0.14). The number of deaths due to hypoglycaemia and cases with unexplained cause of death also remained constant (test for trend: p = 0.44). Chronic alcohol abuse or acute alcohol intoxication was found in 50% of the 135 patients dying from hypoglycaemia, ketoacidosis or unknown cause of death (including found dead in bed), while this was the case in only 16% of the remaining 91 cases dying from other natural causes. We conclude that introduction of human insulin in Denmark was not followed by an increase in sudden deaths among younger insulin-treated diabetic patients.     

Natriuresis after cardiopulmonary bypass: relationship to urodilatin, atrial natriuretic factor, antidiuretic hormone, and aldosterone

OBJECTIVE: Recent studies suggest that urodilatin from the kidneys rather than atrial natriuretic factor from the heart is the more important member of the family of natriuretic peptides involved in the normal regulation of renal sodium and water excretion. We thus examined the relationship between natriuresis, urodilatin, and atrial natriuretic factor in patients after cardiopulmonary bypass, a procedure known to increase levels of atrial natriuretic factor significantly. METHODS: Excretion rates of sodium and water were correlated with the excretion of urodilatin and with circulating levels of atrial natriuretic factor, antidiuretic hormone, aldosterone, and plasma renin activity during a period of 16 hours in 12 patients having had coronary artery bypass operations and with approximately a 400% elevation in levels of atrial natriuretic factor. RESULTS: Natriuresis did not correlate with atrial natriuretic factor, antidiuretic hormone, aldosterone, or plasma renin activity. Excretion rates of urodilatin, however, correlated significantly with excretion rates of sodium (r = 0.74, p = 0.03), urine flow (r = 0.83, p = 0.01), and with levels of serum sodium (r = 0.82, p = 0.01). CONCLUSION: These results suggest an important role for urodilatin, greater than that of atrial natriuretic factor, in the regulation of renal excretion of sodium and water after cardiopulmonary bypass surgery.     

Cyclic guanosine monophosphate responses to atrial natriuretic factor, brain natriuretic peptide, but not C-type natriuretic peptide, and the characterization of their receptors in rat medullary thick ascending limb

The effects of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on renal medullary thick ascending limb (mTAL) have not been fully understood. The aim of this study is to examine the second-messenger responses of rat mTAL to ANF, BNP, and CNP. Characterizations of the ANF, BNP, and CNP receptors in mTAL were also performed by radioligand studies. Results showed that ANF and BNP were both capable of eliciting cyclic guanosine monophosphate (cGMP) responses in mTAL. Conversely, no cGMP response was observed upon stimulation by CNP in mTAL. The presence of ANF receptors was demonstrated by radioligand studies. One receptor site was found, and the Kd and maximum binding capacity were 4.0 +/- 0.45 nmol/L and 277.8 +/- 47.7 fmol/mg protein, respectively. BNP receptors were also found in mTAL, and ANF and BNP were sharing the same receptor. On the contrary, no CNP receptor could be shown by radioligand studies. These results suggest that guanylyl cyclase-coupled receptors (atrial natriuretic peptide receptor-A [ANPR-A]) specific for ANF and BNP are present in rat mTAL, while those for CNP (ANPR-B) are absent. ANF and BNP but not CNP act on mTAL to control water excretion.     

Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi

The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body weight regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.     

Upregulation of angiotensin converting enzyme by atrial natriuretic peptide and cyclic GMP in human endothelial cells

OBJECTIVE: To examine the role of atrial natriuretic peptide (ANP) and cyclic GMP in the regulation of angiotensin converting enzyme (ACE) in cultured human endothelial cells. METHODS: Cultured endothelial cells from human umbilical veins (HUVEC) were treated with ANP (0.3-30 nM), 8-Br-cGMP (1-100 microM), Rp-8-Br-PET-cGMPS (1 microM), or the phosphodiesterase inhibitors, zaprinast (10-100 microM), dipyridamole (1-10 microM), or isobutyl methyl xanthine (IBMX, 0.1-0.5 mM). ACE amounts were measured by inhibitor binding assay and cellular cGMP levels by radioimmunoassay. RESULTS: ANP caused a dose dependent increase in ACE measured in intact endothelial cell culture. The stimulatory effect of ANP was blocked by Rp-8-Br-PET-cGMPS, a protein kinase G inhibitor. The cyclic GMP analog, 8-Br-cGMP and the cyclic GMP specific phosphodiesterase inhibitor, zaprinast, both increased ACE. Increase of ACE was also caused by nonspecific phosphodiesterase inhibitors, dipyridamole and IBMX. Intracellular cGMP levels were shown to increase by ANP, and phosphodiesterase inhibitors. CONCLUSIONS: These data suggest that cGMP is an intracellular mediator regulating ACE and that ANP induced increase of ACE is mediated via a cGMP dependent mechanism.     

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.     

Urocortin, a newly identified corticotropin-releasing factor-related mammalian peptide, stimulates atrial natriuretic peptide and brain natriuretic peptide secretions from neonatal rat cardiomyocytes

Vanadate stimulates adipocyte 2-deoxyglucose transport and GLUT-4 translocation to the membrane through an insulin receptor-independent but wortmannin-inhibitable pathway. Vanadate stimulates PI 3-kinase in anti-IRS-1 immunoprecipitates and the binding between IRS-1 and the p85alpha subunit of PI 3-kinase. In insulin-resistant adipocytes from old rats vanadate fully stimulates IRS-1-associated PI 3-kinase, but partially activates glucose uptake. We conclude that: (a) vanadate stimulates 2-deoxyglucose uptake using a pathway that converges with that of insulin at the level of PI 3-kinase; and (b) adipocytes from old rats are defective in the insulin pathway at steps located both upstream and downstream of PI 3-kinase.     

Refractory ascites in cirrhosis: roles of volume expansion and plasma atrial natriuretic factor level elevation

Cirrhotic patients with ascites refractory to diuretics also have blunted response to marked elevations of plasma atrial natriuretic factor levels alone or to moderate intravascular volume expansion by head-out water immersion. However, these patients usually undergo natriuresis after peritoneovenous shunting. To dissect the factors responsible for this response, we studied the effects on separate days of moderate intravascular volume expansion and highly elevated plasma atrial natriuretic factor levels (head-out water immersion and atrial natriuretic factor infusion) or marked volume expansion and moderate plasma atrial natriuretic factor level elevation (head-out water immersion and albumin infusion) in 13 alcoholic cirrhotic patients with massive ascites. Three of these patients, who responded to initial head-out water immersion with a negative sodium balance, served as controls. Unresponsiveness to head-out water immersion was confirmed in the remaining 10 patients on both days on the basis of blunted natriuretic response (urinary sodium excretion < 0.8 mmol/hr after 2 hr). In contrast, these 10 refractory patients were able to achieve negative sodium balance with both combinations. Mean urinary sodium excretion increased from a baseline level of 0.13 +/- 0.10 mmol/hr to a peak level of 2.29 +/- 0.61 mmol/hr after head-out water immersion and atrial natriuretic factor infusion and from 0.10 +/- 0.3 mmol/hr to 1.61 +/- 0.62 mmol/hr after head-out water immersion and albumin infusion. Both maneuvers were associated with suppression of plasma renin activity and serum aldosterone levels. With head-out water immersion and atrial natriuretic factor infusion, we noted a significant increase in 5' cyclic GMP levels, a second messenger of atrial natriuretic factor, indicating possible activation of atrial natriuretic factor receptors at the inner medullary collecting ducts. In contrast, with head-out water immersion and albumin infusion no such increase in levels occurred, indicating that the increase in urinary sodium excretion was mainly due to increased delivery of sodium to the cortical distal nephron, as indicated by a disproportionate increase in urinary potassium excretion. In conclusion, massive (as opposed to moderate) volume expansion or greatly elevated levels of plasma atrial natriuretic factor associated with moderate volume expansion can improve blunted atrial natriuretic factor responsiveness in cirrhotic patients with refractory ascites. This appears to be achieved by way of a marked increase in distal delivery of filtrate in the kidney, with or without activation of distal atrial natriuretic factor receptors in the inner medullary collecting ducts.     

Different secretion patterns of adrenomedullin, brain natriuretic peptide, and atrial natriuretic peptide during exercise in hypertensive and normotensive subjects

The purpose of this study was to investigate the effect of exercise on plasma concentrations of adrenomedullin, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) in patients with essential hypertension (n = 15) and in normotensive controls (n = 10). Exercise consisted of two fixed workloads, 40 and 80 watts of work load using a supine bicycle ergometer. Plasma levels of all three peptides at rest were significantly higher in hypertensives than in controls. Plasma concentrations of ANP increased with exercise in both groups and had greater increments in hypertensive patients than in normotensives. Plasma concentrations of BNP increased only in patients with hypertension and the levels of increase correlated with basal plasma BNP levels (r = 0.94, p < 0.001) and with left ventricular mass (r = 0.62, p < 0.01) determined by echocardiography. In contrast, plasma adrenomedullin did not change with exercise in either group. These results suggest that secretion patterns of these peptides are regulated by different mechanisms and that the amount and kind of peptides mobilized by exercise may depend on the underlying diseases or pathophysiologic condition.     

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.     

Influence of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor antagonism on renal sodium and water handling and albuminuria during infusion of atrial natriuretic factor into healthy volunteers

Ultrastructural immunocytochemical studies were performed on sections of bone marrow from three patients with beta-thalassaemia major and two patients with haemoglobin H (HbH) disease. Some sections were reacted with either a polyclonal or a monoclonal anti-human-ubiquitin antibody and the reaction visualized using a gold-labelled secondary antibody. The inclusions of precipitated globin chains found within the erythropoietic cells of all five patients reacted much more strongly than the surrounding inclusion-free cytoplasm with both of the anti-ubiquitin antibodies, indicating that the precipitated globin chains were ubiquitinated. A non-specific reaction between the anti-ubiquitin antibodies and the inclusions was excluded by demonstrating that various other antibodies, including a polyclonal anti-human cathepsin D antibody, did not react with the inclusions. The data suggest that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in alpha- and beta-thalassaemia.