Molecular evidence of sustained urban malaria transmission in Amazonian Brazil, 2014-2015.

The relative contribution of imported vs. locally acquired infections to urban malaria burden remains largely unexplored in Latin America, the most urbanised region in the developing world. Here we use a simple molecular epidemiology framework to examine the transmission dynamics of Plasmodium vivax in Mâncio Lima, the Amazonian municipality with the highest malaria incidence rate in Brazil. We prospectively genotyped 177 P. vivax infections diagnosed in urban residents between June 2014 and July 2015 and showed that local parasites are structured into several lineages of closely related microsatellite haplotypes, with the largest genetic cluster comprising 32% of all infections. These findings are very unlikely under the hypothesis of multiple independent imports of parasite strains from the rural surroundings. Instead, the presence of an endemic near-clonal parasite lineage circulating over 13 consecutive months is consistent with a local P. vivax transmission chain in the town, with major implications for malaria elimination efforts in this and similar urban environments across the Amazon.

High levels of IgM against methylglyoxal-modified apolipoprotein B100 are associated with less coronary artery calcification in patients with type 2 diabetes.

OBJECTIVE: Advanced glycation end products (AGE) have been implicated in diabetic vascular complications through activation of pro-inflammatory genes. AGE-modified proteins are also targeted by the immune system resulting in the generation of AGE-specific autoantibodies, but the association of these immune responses with diabetic vasculopathy remains to be fully elucidated. The aim of this study was to determine whether antibodies against apolipoprotein B100 modified by methylglyoxal (MGO-apoB100) are associated with coronary atherosclerosis in patients with type 2 diabetes. METHODS: We measured antibodies against MGO-apoB100 in plasma from 497 type 2 diabetic patients without clinical signs of cardiovascular disease. Severity of coronary disease was assessed as coronary artery calcium (CAC) imaging. Immunoglobulin (Ig)M and IgG levels recognizing MGO-apoB100 were determined by enzyme-linked immunosorbent assay. RESULTS: Anti-MGO-apoB100 IgM antibody levels were higher in subjects with a low to moderate CAC score (≤400 Agatston units) than in subjects with a high score (>400 Agatston units; 136.8±4.4 vs. 101.6± 7.4 arbitrary units (AU), P<0.0001) and in subjects demonstrating no progression of CAC during 30 months of follow-up (136.4±5.7 vs. 113.9 ± 6.2 AU in subjects with progression, P<0.0001). Subjects with a family history of premature myocardial infarction had lower levels of anti-MGO-apoB100 IgM. Female subjects had higher levels of anti-MGO-apoB100 antibodies and lower CAC than men. Accordingly, high levels of IgM against MGO-apoB100 are associated with less severe and a lower risk of progression of coronary disease in subjects with type 2 diabetes. CONCLUSIONS: Although conclusions regarding causal relationships based on epidemiological observations need to be made with caution, our findings suggest the possibility that anti-MGO-apoB100 IgM may be protective in diabetic vasculopathy.

Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Oxidation of LDL in the arterial extracellular matrix is a key event in the development of atherosclerosis and autoantibodies against oxidised LDL antigens reflect disease severity and the risk of developing acute cardiovascular events. Since type 2 diabetes is associated with increased oxidative stress, we tested the hypothesis that autoantibodies against oxidised LDL antigens are biomarkers for vascular complications in diabetes. METHODS: We studied 497 patients with type 2 diabetes without clinical signs of coronary heart disease. Oxidised LDL autoantibodies were determined by ELISA detecting IgG and IgM specific for native and malondialdehyde (MDA)-modified apolipoprotein B-100 peptides p45 and p210. The severity of coronary disease was assessed as the coronary artery calcium score. RESULTS: Patients affected by retinopathy had significantly higher levels of IgG against MDA-p45 and MDA-p210. In contrast, high levels of autoantibodies against the corresponding native peptides were associated with less coronary calcification and a lower risk of progression of coronary disease. CONCLUSIONS/INTERPRETATION: Our observations suggest that LDL oxidation is involved in the pathogenesis of diabetic retinopathy and that autoantibodies against apolipoprotein B peptides may act as biomarkers for both micro- and macrovascular complications in diabetes.

Oenology: red wine procyanidins and vascular health.

Regular, moderate consumption of red wine is linked to a reduced risk of coronary heart disease and to lower overall mortality, but the relative contribution of wine's alcohol and polyphenol components to these effects is unclear. Here we identify procyanidins as the principal vasoactive polyphenols in red wine and show that they are present at higher concentrations in wines from areas of southwestern France and Sardinia, where traditional production methods ensure that these compounds are efficiently extracted during vinification. These regions also happen to be associated with increased longevity in the population.

Effects of tumour necrosis factor-alpha in the human forearm: blood flow and endothelin-1 release.

Increased circulating concentrations of tumour necrosis factor-alpha (TNF-alpha) are seen in several pathological conditions associated with vascular disease. TNF-alpha induces the synthesis of endothelin-1 (ET-1), a potent vasoconstictor, by the endothelium. However, there is profound vasodilatation in sepsis, where circulating levels of both ET-1 and TNF-alpha are elevated. The details of the interaction between ET-1 and TNF-alpha and the predominant resulting haemodynamic effect in healthy humans are unclear. The aim of the present study was to determine the effects of intra-arterial TNF-alpha on ET-1 spillover, vascular tone and endothelial function in the healthy human forearm. Brachial arterial and deep venous blood samples, forearm plasma flow measurements and blood flow responses to acetylcholine and sodium nitroprusside were obtained in six healthy subjects before and during a 6 h infusion of TNF-alpha into the brachial artery. Forearm blood flow was significantly greater than baseline during exposure to TNF-alpha [median (lower quartile, upper quartile): baseline, 2.6 (2.1, 2.8) ml.min(-1).100 ml(-1); TNF-alpha, 4.6 (4.5, 5.1) ml.min(-1).100 ml(-1); P <0.05]. The rate of release of ET-1 was significantly greater than baseline after 30 and 260 min of TNF-alpha infusion [median (lower quartile, upper quartile): baseline, 0.8 (0.6, 1.1) pg.min(-1).100 ml(-1); 30 min, 2.4 (1.9, 3.2) pg.min(-1).100 ml(-1); 260 min, 4.1 (3.1, 4.2) pg.min(-1).100 ml(-1); P <0.05]. The vasodilatory response to acetylcholine was diminished during TNF-alpha infusion, whereas the response to sodium nitroprusside remained unchanged. We thus demonstrate for the first time that local TNF-alpha increases ET-1 spillover from the human forearm and impairs endothelium-dependent vasodilatation. In spite of this action, TNF-alpha has a vasodilatory effect, resulting in an increase in forearm blood flow.

Oxidative stress participates in the breakdown of neuronal phenotype in experimental diabetic neuropathy.

AIMS/HYPOTHESIS: This study compared the effects of streptozotocin-induced diabetes in rats with those of two pro-oxidant interventions; a diet deficient in vitamin E and treatment with primaquine. METHODS: Measurements were made by the classic motor and sensory conduction velocity deficits and by indicators of the breakdown of small fibre phenotype i.e., sciatic nerve content of nerve growth factor and the neuropeptides, substance P and neuropeptide Y. RESULTS: As with diabetes, the pro-oxidant interventions decreased conduction velocities (though the effect of vitamin E deficiency was not significant), the sciatic nerve content of nerve growth factor and the neuropeptides (all percentages refer to the mean value for the appropriate control groups). In diabetes, nerve growth factor was depleted to 50% in the control rats (p < 0.05); oxidative stress depleted nerve growth factor to 64% (primaquine; p < 0.05) and 81% (vitamin E deficient; not significant) of controls. Substance P was depleted to 51% in the control rats (p < 0.01) with depletions to 74% and 72% (both p < 0.01) by oxidative stress; equivalent depletions for neuropeptide Y were 38% controls in diabetes (p < 0.001) and 67% (primaquine; p < 0.001) and 74% (vitamin E deficient; p < 0.05) for oxidative stress. CONCLUSION/INTERPRETATION: The relative magnitudes of these changes suggest an effect in diabetes of oxidative stress, coupled with some other cellular event(s). This is supported by the effects of a diester of gamma-linolenic acid and alpha-lipoic acid, which completely prevented the effects on the pro-oxidant interventions on conduction velocity, nerve growth factor and neuropeptide contents, but was only partially preventative in diabetes.

Role of hypothalamic neuropeptide Y and orexigenic peptides in anorexia associated with experimental colitis in the rat.

Neuropeptide Y (NPY) is thought to play a crucial role in the normal hypothalamic response to starvation. After a period of food restriction, increased release of NPY induces hunger and hyperphagia, and helps to restore body weight to its set point. Persistent anorexia in rats with experimental colitis implies failure of this adaptive feeding response. In vivo NPY release and regional hypothalamic NPY concentrations were measured in rats with trinitrobenzenesulphonic acid (TNBS)-induced colitis, healthy controls and animals pair-fed to match the food intake of the colitic group. Food intake in the colitic group was assessed after administration of NPY and two other potent orexigenic peptides: melanin-concentrating hormone (MCH) and hypocretin (orexin-A). Food intake was decreased by 30-80% below control values for 5 days in the colitic rats. In both the pair-fed and colitic groups, release of NPY in the paraventricular nucleus was significantly increased compared with free-feeding controls. Intraventricular or intrahypothalamic administration of NPY, MCH or hypocretin elicited a feeding response in healthy controls, but not in the colitic group. In summary, animals with TNBS-colitis and anorexia show an appropriate increase in hypothalamic NPYergic activity. However, the failure of NPY and other orexigenic peptides to increase feeding in the colitic group indicates suppression of feeding, either by inhibition of a common downstream hypothalamic neuronal pathway or by induction of one or more potent anorexigenic agents.

Effect of an antisense oligodeoxynucleotide to endothelin-converting enzyme-1c (ECE-1c) on ECE-1c mRNA, ECE-1 protein and endothelin-1 synthesis in bovine pulmonary artery smooth muscle cells.

Endothelin-1 (ET-1) is secreted from endothelial and vascular smooth muscle cells (VSMC) after intracellular hydrolysis of big ET-1 by endothelin converting enzyme (ECE). The metallopeptidase called ECE-1 is widely thought to be the physiological ECE, but unequivocal evidence of this role has yet to be provided. Endothelial cells express four isoforms of ECE-1 (ECE-1a, ECE-1b, ECE-1c, and ECE-1d), but the identity of ECE-1 isoforms expressed in VSMC is less clear. Here, we describe the characterization of ECE-1 isoforms in bovine pulmonary artery smooth muscle cells (BPASMC) and the effect on ET-1 synthesis of an antisense oligodeoxynucleotide (ODN) to ECE-1c. Reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation of total RNA from BPASMC showed that ECE-1a and ECE-1d were not expressed. Sequencing of cloned ECE-1 cDNA products and semiquantitative RT-PCR demonstrated that ECE-1b and ECE-1c were expressed in BPASMC, with ECE-1c being the predominant isoform. Basal release of ET-1 from BPASMC was low. Treatment for 24 h with tumor necrosis factor-alpha (TNFalpha) stimulated ET-1 production by up to 10-fold with parallel increases in levels of preproET-1 mRNA. Levels of ECE-1c mRNA were also raised after TNFalpha, whereas amounts of ECE-1b mRNA were decreased significantly. Treatment of BPASMC with a phosphorothioate antisense ODN to ECE-1c caused a marked reduction in ECE-1c mRNA levels and ECE-1 protein levels. However, basal and TNFalpha-stimulated ET-1 release were largely unaffected by the ECE-1c antisense ODN despite the inhibition of ECE-1c synthesis. Hence, an endopeptidase distinct from ECE-1 is mainly responsible big ET-1 processing in BPASMC.

Endothelin-1 synthesis reduced by red wine.

Statistical evidence of reduced coronary heart disease in areas of high wine consumption has led to the widespread belief that wine affords a protective effect. Although moderate drinking of any alcohol helps to reduce the incidence of coronary heart disease, there is no clear evidence that red wine confers an additional benefit. Here we show that red wines strongly inhibit the synthesis of endothelin-1, a vasoactive peptide that is crucial in the development of coronary atherosclerosis. Our findings indicate that components specific to red wine may help to prevent coronary heart disease.

Relationship between soluble intracellular endothelin-converting enzyme and endothelin-1 synthesis: effect of inhibitors of the secretory pathway.

The relationship between soluble and membrane-bound endothelin-converting enzyme (ECE) activity with the level of endothelin-1 (ET-1) synthesis was investigated in cultured endothelial cells. Escherichia coli lipopolysaccharide (LPS) was used to stimulate ET-1 synthesis, and brefeldin A, monensin, colchicine or cytochalasin B, which disrupt peptide biosynthetic pathways in a variety of ways, were tested for their ability to modify changes in ET-1 synthesis and ECE levels. LPS increased ET-1 secretion by more than twofold. Levels of soluble ECE activity, but not those of membrane-bound ECE activity, correlated with ET-1 synthesis. These results suggest the soluble ECE activity is likely to play a role in ET-1 biosynthesis.

Effect of lisinopril on tissue levels of neuropeptide Y in normotensive and spontaneously hypertensive rats.

Angiotensin-converting enzyme (ACE) inhibitors reduce systemic and coronary vasoconstriction by modulating sympathetic neuroeffector function and by decreasing sympathetic activation. Here, blood pressure, and tissue concentrations of noradrenaline and neuropeptide Y (NPY) were studied in normotensive and spontaneously hypertensive rats (SHR) after 2 weeks treatment with lisinopril (0.3 mg/day; osmotic mini-pump). MAP was reduced in both normotensive rats and SHR after lisinopril by 32 mm Hg and 66 mm Hg respectively (P < 0.001 compared to corresponding control rats). NPY levels were significantly higher in extracts of atria, kidney, spleen and adrenal of normotensive rats compared to SHR. Lisinopril treatment increased NPY levels in atria and skeletal muscle extracts of SHR by 15% and 70% respectively (P < 0.05). Lisinopril also significantly increased noradrenaline content of the atria by 16% in SHR (P < 0. 05). The decrease in MAP and increase in tissue levels of sympathetic neurotransmitters provide further evidence that inhibition of ACE decreases sympathetic neurotransmission leading to accumulation of stored neurotransmitters. Journal of Human Hypertension (2000) 14, 381-384

Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression.

ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor-alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.

The p55 tumor necrosis factor receptor (CD120a) induces endothelin-1 synthesis in endothelial and epithelial cells.

Synthesis of the vasoconstrictor peptide endothelin-1 by endothelial and epithelial cells is strongly induced by tumor necrosis factor alpha (TNF-alpha). The actions of TNF-alpha are mediated by two transmembrane receptors of approximately 55 (p55, CD120a) and 75 kDa (p75, CD120b). Reagents activating selectively these receptor subtypes have been used to identify which TNF receptor mediates the induction of endothelin-1 synthesis. Stimulation of bovine aortic endothelial cells or human HEp-2 epithelial cells with a p55-selective mutant of human TNF-alpha (R32W-S86T) induced significant and concentration-dependent increases in endothelin-1 release. A p75 receptor-selective TNF-alpha mutant (D143N-A145R) was ineffective alone or in combination with the p55-selective mutant. Competitive binding experiments with [125I]TNF-alpha showed the p55-selective mutant, but not the p75-selective mutant, to inhibit the binding of [125I]TNF-alpha to endothelial and HEp-2 cells. Similar results were obtained with the p55 agonist antibody htr1 in both cell lines. These results establish the p55 TNF receptor as the main receptor involved in the induction of endothelin-1 synthesis by TNF-alpha.

The expression of endothelin-1 and endothelin-converting enzyme-1 (ECE-1) are independently regulated in bovine aortic endothelial cells.

The endopeptidase called endothelin-converting enzyme-1 (ECE-1) is thought to play a physiological role in endothelin-1 (ET-1) biosynthesis. For human ECE-1, differential splicing of messenger RNA (mRNA) results in the synthesis of three isoforms, termed ECE-1a, ECE-1b, and ECE-1c. The isoform(s) responsible for the hydrolysis of the biosynthetic intermediate big ET-1 in endothelial cells have yet to be assigned. To investigate whether the expression of mRNAs for preproET-1 and ECE-1 are regulated in parallel, a variety of conditions were used to compare levels of ET-1 synthesis by bovine aortic endothelial cells (BAECs) with levels of mRNA for preproET-1, ECE-1a, ECE-1c, and the combined ECE-1 isoforms (ECE-1a/b/c). Stimulation of BAECs with tumor necrosis factor-alpha or transforming growth factor-beta increased ET-1 synthesis, and treatment of BAECs with 2-chloroadenosine or staurosporine caused concentration-dependent reductions in ET-1 synthesis. Estimates of mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR) with linear cycling conditions showed changes in preproET-1 expression to correlate well with ET-1 secretion. In contrast, RT-PCR analysis of ECE-1 expression by using primer pairs to measure ECE-1a, ECE-1c, or all the ECE-1 isoforms simultaneously showed no correlation between their mRNA levels and those of preproET-1. This indicates that under the conditions investigated, expression of ECE-1 is not coordinated with ET-1 synthesis in BAECs.

Studies of the role of endothelium-dependent nitric oxide release in the sustained vasodilator effects of corticotrophin releasing factor and sauvagine.

1. The mechanisms of the sustained vasodilator actions of corticotrophin-releasing factor (CRF) and sauvagine (SVG) were studied using rings of endothelium de-nuded rat thoracic aorta (RTA) and the isolated perfused rat superior mesenteric arterial vasculature (SMA). 2. SVG was approximately 50 fold more potent than CRF on RTA (EC40: 0.9 +/- 0.2 and 44 +/- 9 nM respectively, P < 0.05), and approximately 10 fold more active in the perfused SMA (ED40: 0.05 +/- 0.02 and 0.6 +/- 0.1 nmol respectively, P < 0.05). Single bolus injections of CRF (100 pmol) or SVG (15 pmol) in the perfused SMA caused reductions in perfusion pressure of 23 +/- 1 and 24 +/- 2% that lasted more than 20 min. 3. Removal of the endothelium in the perfused SMA with deoxycholic acid attenuated the vasodilatation and revealed two phases to the response; a short lasting direct action, and a sustained phase which was fully inhibited. 4. Inhibition of nitric oxide synthase with L-NAME (100 microM) L-NMMA (100 microM) or 2-ethyl-2-thiopseudourea (ETPU, 100 microM) had similar effects on the vasodilator responses to CRF as removal of the endothelium, suggesting a pivotal role for nitric oxide. However the selective guanylate cyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 10 microM) did not affect the response to CRF. 5. High potassium (60 mM) completely inhibited the vasodilator response to CRF in the perfused SMA, indicating a role for K channels in this response. 6. Compared to other vasodilator agents acting via the release of NO, the actions of CRF and SVG are strikingly long-lasting, suggesting a novel mechanism of prolonged activation of nitric oxide synthase.

Corticotrophin releasing factor attenuates NG-nitro-L-arginine methyl ester induced increases in blood pressure in the anaesthetised rat.

In vitro corticotrophin releasing factor (CRF) causes a prolonged endothelium-dependent vasodilatation which is blocked by inhibitors of nitric oxide (NO) synthase (NOS). Here the role of NO in CRF-induced reductions in blood pressure (BP) has been investigated using anaesthetised rats. Infusion of NG-Nitro-L-arginine Methyl Ester (L-NAME) increased mean arterial pressure (MAP) but did not reduce the fall in BP caused by CRF injection. Bolus injection of L-NAME produced a marked vasopressor effect when given alone. However, when administered 20 min after CRF, L-NAME restored MAP to basal levels, but the marked vasopressor response observed in control animals was completely blocked. This indicates that the increase in BP occurring on bolus injection of L-NAME is not simply due to an inhibition of NO-dependent vasodilatation.

A lipoic acid-gamma linolenic acid conjugate is effective against multiple indices of experimental diabetic neuropathy.

Untreated streptozotocin-diabetic (7 weeks duration) rats showed reductions (all p < 0.01; percentages in brackets) in motor and sensory nerve conduction velocity (MNCV; 14%, SNCV; 17%) and in sciatic nerve contents of nerve growth factor (NGF; 57%), substance P (SP; 53%) and neuropeptide Y (NPY; 39%). Treatment with a gamma-linolenic acid-alpha-lipoic acid conjugate (GLA-LA; 35 mg x day(-1) x rat(-1)) attenuated (p < 0.05) these reductions to MNCV (8%), SNCV (5%), NGF (19%), SP (23%), NPY (20%), such that the values in GLA-LA-treated diabetic rats did not differ significantly from those of control non-diabetic animals. Treatment with alpha-lipoic acid alone at 100 mg/kg i.p. was without effect on these variables except for NGF (33% reduction, p < 0.05) and treatment with the antioxidant, butylated hydroxytoluene (1.5% dietary supplement) did not affect any deficits. These data show that GLA-LA is effective in improving both electrophysiological and neurochemical correlates of experimental diabetic neuropathy.

Cloning of bovine preproadrenomedullin and inhibition of its basal expression in vascular endothelial cells by staurosporine.

The cDNA encoding preproadrenomedullin (preproAM) was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends from total RNA from bovine aortic endothelial cells (BAEC). Bovine preproAM cDNA shows high sequence homology with human, porcine and rat preproAM. Bovine-specific primers derived from this sequence were used in RT-PCR to study regulation of this gene. Treatment of BAEC or a human endothelial cell line (Ea.hy 926) with the non-selective protein kinase inhibitor staurosporine resulted in significantly reduced preproAM mRNA levels. The reduction in preproAM mRNA appeared to be absolute when Ea.hy 926 cells were exposed to 100 nM staurosporine for 2 h. However, this dramatic reduction could not be reproduced by treatment with the protein kinase A (PKA) inhibitor H-89, or the protein kinase C (PKC) inhibitors chelerythrine chloride and bisindolylmaleimide I. These observations suggest that activation of a novel staurosporine-sensitive protein kinase is necessary for basal expression of the preproAM gene in these cells.

Studies of endothelin-converting enzyme in bovine endothelial cells and vascular smooth-muscle cells: further characterization of the biosynthetic process.

Endothelin-1 (ET-1) is synthesized by a number of cell types, including endothelial, epithelial, and smooth muscle cells. Initial biosynthesis occurs as a protein precursor, preproendothelin-1 (preproET-1). This is processed intracellularly to the inactive intermediate big ET-1, which is hydrolyzed by endothelin-converting enzyme (ECE) to generate ET-1, but the precise identity of the physiologically relevant ECE has yet to be confirmed. Although ET-1 is synthesized in the constitutive secretory pathway, many features of the selective processing of proET-1 are comparable to those of peptide hormones. We describe here experimental investigations aimed at defining the regulation of ET-1 synthesis and its relationship to the biosynthesis of ECE.

Comparison of the regulation of endothelin-2 and endothelin-converting enzyme-1 b [correction of beta] by forskolin and TNF-alpha in ACHN cells.

The effects on the expression and secretion of ET-2 of forskolin and tumor necrosis factor-alpha (TNF-alpha) were investigated using the human renal adenocarcinoma (ACHN) cell line. For comparison, changes in endothelin-converting enzyme-1 b (ECE-1 b) [corrected] mRNA were also determined. Treatment for 4 with TNF-alpha (3 ng/ml), forskolin (30 microM), or the combination caused significant increases in ET-2 release, TNF-alpha alone or in combination with forskolin increased ET-2 mRNA levels at 1 h and 2 h. After 4 h the expression of ET-2 mRNA was comparable to control levels. In contrast to ET-2, ECE-1 b [corrected] mRNA levels were increased by TNF-alpha only at 4 h. Forskolin increased expression of ET-2 mRNA at 1 and 4 but had no significant effect on expression of ECE-beta. These data suggest that expression of ET-2 and ECE-1 b [corrected] mRNA is regulated differently in ACHN cells.