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.

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.

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.

Dexamethasone treatment in man induces changes in 24-hour growth hormone (GH) secretion profile without altering total GH released.

Glucocorticoids inhibit growth in man and laboratory animals and reduce the GH response to the majority of exogenously administered stimuli. Recently, however, glucocorticoids have been shown to have varying effects on GH secretion depending on the time of administration and, furthermore, to be potent secretagogues in their own right. To investigate this further, we have carried out sampling over two 24-h periods in six normal male volunteers both before and directly after treatment with dexamethasone (DEX; 2 mg twice daily) for 96 h. After DEX administration, all volunteers showed an increase in mean GH secretion during the first 9 h of sampling (0900-1800 h) compared with pre-DEX control profiles (5.1 +/- 1.2 vs. 1.7 +/- 0.5 micrograms/L; P less than 0.001). DEX treatment also had the effect of delaying and attenuating the nocturnal peak; mean GH secretion between 0000-0200 h was significantly greater before DEX (13.6 +/- 2.7 micrograms/L) than after DEX (3.6 +/- 0.7 micrograms/L; P less than 0.001), whereas that between 0300-0800 h was greater after DEX (5.5 +/- 0.8 vs. 0.7 +/- 0.2 micrograms/L; P less than 0.001). Individual nocturnal peaks ranged from 7.0-56.8 micrograms/L, occurring between 0030-0200 h before DEX, and 2.6-21.2 micrograms/L, occurring between 0300-0400 h after DEX. Overall mean GH secretion was not significantly altered by DEX treatment (3.8 +/- 0.6 vs. 4.2 +/- 0.5 micrograms/L; P = NS). Total insulin-like growth factor-I (IGF-I) levels, measured after acid-ethanol extraction, were significantly increased by DEX treatment, with mean IGF-I over the 24-h sampling period rising from 292.2 +/- 31.8 to 425.9 +/- 37 micrograms/L (P less than 0.005). All individuals showed an increase in mean 24-h IGF-I of between 10-75%. In a second study, 12 male volunteers were treated with DEX in an identical manner, and blood was taken at 0800 h daily. Total IGF-I levels rose steadily from 307.9 +/- 13.3 micrograms/L, reached a plateau at 72 h and remained elevated at 96 h (424.9 +/- 16.5 micrograms/L; P less than 0.001). These results suggest that glucocorticoids alter the normal pattern of GH secretion with an increase in daytime levels, but a delaying and attenuating effect on the nocturnal pulse. Previous studies have suggested that IGF-I concentrations are decreased by steroid treatment, but these have been based on bioassay systems. Total IGF-I, measured by RIA, would appear to be consistently elevated; the apparent decrease seen in bioassay systems may be due to glucocorticoid-induced changes in binding protein concentrations.

Effects of dexamethasone on growth hormone (GH)-releasing hormone, arginine- and dopaminergic stimulated GH secretion, and total plasma insulin-like growth factor-I concentrations in normal male volunteers.

In man, glucocorticoid treatment and endogenous corticosteroid excess generally suppress stimulated GH release. However, such effects are not entirely consistent and depend on both the duration of pituitary exposure to steroids and the secretagogue employed. To further evaluate the effects of glucocorticoids in man, we have studied the response to four different GH stimulation tests before and after treatment with dexamethasone (DEX; 2 mg twice daily during 84 h). Twelve healthy male volunteers were divided into two groups of six subjects (groups A and B). Group A underwent stimulation tests with arginine (500 mg/kg, i.v.) and GH-releasing hormone (GHRH, 100 micrograms, i.v.) before and after DEX treatment. Group B were subjected to stimulation tests with two dopaminergic agents, a novel nonergot D2-dopamine agonist CV205-502 (CV; 10 micrograms, i.v.) and dopamine (4 micrograms/kg.min, i.v.), before and after DEX. Within each group, the effect of DEX on the different secretagogues was studied 4 weeks apart. GHRH-stimulated GH release was significantly blunted by DEX treatment [median peak GH value, 34.2 micrograms/L; 25-75th percentiles, 22.1-56.2), control, vs. 19.8 (9.7-34.5), DEX; P less than 0.05; integrated GH secretion expressed as the area under the curve (AUC) was 48% lower after DEX; P less than 0.01]. In the same group, DEX treatment significantly enhanced the response to arginine [10.6 (8.0-22.8), control, vs 26.1 (15.1-38.6), DEX; P less than 0.01; with an increase in AUC of 72%; P less than 0.01]. In group B, under control conditions before glucocorticoid administration, the GH response to CV was significantly greater than that to dopamine in terms of both peak response [25.1 (8.6-30.9), CV, vs. 11.8 (5.5-16.4), dopamine; P less than 0.05] and AUC [2406 +/- 654 (CV) vs. 658 +/- 125 (dopamine); P less than 0.01], suggesting that CV may be a useful adjunct in the diagnosis of GH deficiency. After DEX administration, responses to both dopaminergic agents were suppressed [CV, 6.7 (4.0-21.2); P less than 0.01 vs. control response; and dopamine, 5.3 (4.8-7.9); P less than 0.05 vs. control response]. When compared with the effects of dexamethasone on the GH response to arginine, the results with dopaminergic agents highlight important differences in the mechanisms of action of these indirectly acting GH secretagogues. Moreover, this may be of physiological importance, because in contrast to the inhibitory effect of glucocorticoid on GHRH-stimulated GH release, DEX treatment significantly increased basal plasma GH levels [1.4 (0.5-5.1) vs. control 0.3 (0.1-0.6) microgram/L; P less than 0.001].(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of growth hormone release in man by the potent D2-dopamine agonist CV 205-502: comparison of responses to intravenous and oral administration.

It is well known that dopaminergic agents are stimulators of GH release in man, and although responses are sometimes unreliable, oral L-dopa and iv dopamine have frequently been employed in the evaluation of GH-deficient states. To assess the effects on GH secretion of a new potent D2-dopamine agonist, the octahydrobenzo(g)quinoline CV 205-502 (CV) we have investigated the GH responses in healthy male volunteers to four different iv doses. For this purpose 3 separate groups of 9 subjects were studied. The respective groups were administered on separate occasions 10 micrograms CV and placebo (group 1), 5 micrograms, 2.5 micrograms, and placebo (group 2), and 1 microgram and placebo (group 3). Each subject received drug and placebo in a double blind randomly assigned order, with at least 5 days between their administration. Active compound or placebo was infused over 30 min, and blood sampling was carried out for 72 h after cessation of infusion. Peak GH levels occurred between 45-60 min after the end of the infusion; the observed maximum GH concentrations were 19.2 +/- 2.9 micrograms/L (10 micrograms, iv; P less than 0.001 vs. placebo), 9.61 +/- 2.1 (5 micrograms, iv; P less than 0.001 vs. placebo), 4.7 +/- 1.7 (2.5 micrograms, iv; P less than 0.05 vs. placebo), and 1.9 +/- 0.8 micrograms/L (1 micrograms, iv; P = NS vs. placebo). The mean integrated GH secretion expressed in arbitrary units [area under the response curve (AUC)] up to 3 h postinfusion showed a typical dose-response relationship. Mean values were 1715 +/- 269.4 (10 micrograms, iv; P less than 0.001 vs. placebo), 956.1 +/- 189.9 (5 micrograms, iv; P less than 0.001 vs. placebo), 312.8 +/- 105.8 (2.5 micrograms, iv), and 162.8 +/- 47.5 (1 microgram, iv). In a second study with a separate group of 18 volunteers, we compared the GH response to an oral dose of 100 micrograms CV with those to 5 micrograms CV given iv and placebo treatment. Peak GH values in this study were 20.3 +/- 5.5 micrograms/L (100 micrograms, orally; P less than 0.01 vs. placebo) and 14.6 +/- 2.8 (5 micrograms, iv; P less than 0.001 vs. placebo). Maximum levels occurred 45 min after the infusion and 90 min after ingestion (60 min relative to the end of the infusion).(ABSTRACT TRUNCATED AT 400 WORDS)

A molecular model for human Big-Endothelin-1 (Big ET-1).

A molecular model has been developed for human Big Endothelin-1, which is the immediate precursor to the potent vasoconstrictor polypeptide endothelin-1 and the target of the highly specific endothelin converting enzyme. This model is produced by a threading algorithm protocol and is consistent with all the currently available structural and biochemical data for this molecule.

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 actions of neuropeptide Y and peptide YY on the hepatic arterial and portal vascular beds of the anaesthetized dog.

1. The vascular actions of the two peptides, neuropeptide Y (NPY) and peptide YY (PYY) were compared with the transmitter noradrenaline (NA) on the arterial and portal vascular beds of the in situ liver of the anaesthetized dog. 2. The sole vascular response of the hepatic arterial vasculature to intra-arterial administration of either NPY or PYY was vasoconstriction; the duration of these responses was longer than that to NA. 3. The maximum hepatic arterial vasoconstrictor responses to PYY and to NPY were significantly different and they were both significantly less than the maximum to NA (P less than 0.001). 4. In contrast to its activity on the splenic arterial vasculature PYY was not more potent, on a molar basis, than NPY as an hepatic arterial vasoconstrictor agent. However, both peptides were significantly more potent than NA (P less than 0.005). 5. Neither peptide, when injected intraportally, caused any change in intrahepatic portal inflow resistance. 6. Both peptides when administered intraportally in doses which were free of systemic effects caused hepatic arterial vasoconstriction.

A comparison of the differential effects of vasoactive intestinal peptide and peptide histidine isoleucine on the vascular and capsular smooth muscle of the dog spleen.

1. The actions of the two peptides, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) have been compared to that of isoprenaline on the smooth muscle systems of the isolated blood-perfused dog spleen. 2. Intra-arterial injections of VIP and PHI caused graded increases in splenic arterial blood flow at constant perfusion pressure indicative of splenic arterial vasodilatation. 3. VIP was significantly more potent than PHI, with their respective molar ED50 values being 9.9 +/- 3.7 and 830 +/- 141 pmol (P less than 0.002). VIP was approximately 10 and 200 times more potent than isoprenaline and PHI respectively. 4. The maximum reduction in splenic arterial vascular resistance was the same (P greater than 0.5) in response to intra-arterial VIP and PHI, although both peptide maxima were significantly less (P less than 0.05, 0.01 respectively) than that obtained with isoprenaline. 5. Small increases in spleen volume accompanied the splenic vasodilator responses to both peptides. They were probably passive in origin, secondary to splenic arterial vasodilatation. 6. The selective beta 2-adrenoceptor antagonist, ICI 118,551, did not antagonize the splenic arterial vasodilator response to VIP or PHI but markedly attenuated the effect of isoprenaline. 7. These observations indicate that VIP and PHI, when either co-released locally or present together in the systemic circulation, may exert a differential action on different components of the circulation.

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.

The actions of the peptides, neuropeptide Y and peptide YY, on the vascular and capsular smooth muscle of the isolated, blood-perfused spleen of the dog.

The two peptides, neuropeptide Y (NPY) and peptide YY (PYY) were compared for potency on the vascular and extravascular smooth muscle of the isolated, blood-perfused spleen of the dog. The only vascular response to both NPY and PYY was vasoconstriction; the maximum effect was to arrest splenic arterial blood flow completely. On a molar basis both NPY and PYY were significantly more potent (P less than 0.01) as splenic arterial vasoconstrictors than the transmitter noradrenaline (NA). PYY was approximately 7 times more potent as a vasoconstrictor than NPY. In contrast to their potency on the vascular smooth muscle, NPY and PYY were significantly (P less than 0.01) less potent than NA in causing contraction of the splenic capsule. The two peptides were equipotent in eliciting this contraction.

Incomplete inhibition of the pressor effects of endothelin-1 and related peptides in the anaesthetized rat with BQ-123 provides evidence for more than one vasoconstrictor receptor.

1. The effects of the ETA receptor antagonist, BQ-123 on blood pressure changes induced by various members of the endothelin (ET)/sarafotoxin (SX) peptide superfamily were investigated in the anaesthetized rat. 2. ET-1 (1 nmol kg-1, i.v. bolus) induced a sustained increase in mean arterial pressure (MAP, maximum increase 44 +/- 3 mmHg). Intravenous injection of BQ-123 at 0.2, 1.0 or 5.0 mg kg-1 5 min before ET-1 inhibited the pressor response by 18, 50 and 61%, respectively. The ET-1 pressor response was inhibited by 75% when the peptide was given 60 min after the start of a 120 min i.v. infusion of BQ-123 (0.2 mg kg-1 min-1). 3. In addition to ET-1, BQ-123 (1 mg kg-1, i.v. bolus) attenuated the pressor responses to big ET-1 (1 nmol kg-1, i.v., bolus, maximum increase in MAP: 68 +/- 7 mmHg), ET-3 (3 nmol kg-1, i.v., bolus, maximum response: 30 +/- 3 mmHg), SX6b (1 nmol kg-1, i.v., bolus, maximum response: 41 +/- 5 mmHg) and SX6c (1 nmol kg-1, i.v., bolus, maximum response: 24 +/- 4 mmHg) by 65, 60, 88 and 50%, respectively. 4. With the exception of big ET-1, all the peptides used in this study induced an initial transient depressor response (-32 +/- 3 mmHg, n = 18). Although BQ-123 (1 mg kg-1, i.v., bolus) did not affect the absolute magnitude of the fall in MAP, the ETA receptor antagonist significantly prolonged the depressor responses induced by ET-3 and SX6b. 5. Thus, BQ-123 attenuates the pressor, but not the depressor effects of ET-1, big ET-1, ET-3, SX6b and SX6c. Complete inhibition of the pressor responses could not be achieved, suggesting that a component of the pressor response is not mediated via the ETA receptor.

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF.

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.

Generation by the phosphoramidon-sensitive peptidases, endopeptidase-24.11 and thermolysin, of endothelin-1 and c-terminal fragment from big endothelin-1.

1. Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24.11 or thermolysin convert big ET-1 to endothelin-1 (ET-1) and C-terminal fragment (CTF), the effects on porcine and human big ET-1 of each of the purified enzymes were compared in vitro. 2. For E-24.11, the relative rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative half-lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET-1 > 24 h; ET-1, 37 min; CTF, 57 min. For comparison, the half-life for hydrolysis of substance P under similar conditions was 2.1 min. 3. For thermolysin the relative rates of hydrolysis were found to be big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET-1, 25 min; ET-1, 56 min; CTF, 47 min. 4. Because the low rate of conversion of big ET-1 to ET-1 by E-24.11 did not yield sufficient ET-1 for h.p.l.c. quantification a RIA specific for ET-1(16-21) was used to study further the hydrolysis of big ET-1 by E-24.11. Incubation of big ET-1 (0.2-2 nmol) with E-24.11 (4-400 ng) generated ET-1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h showed that steady state levels of ET-1 were achieved after 4 h indicating that the rate of ET-1 degradation was then equal to the formation of new ET-1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry.5. Both ET-l-like and CTF-like peaks were detected at 214 nm when the products of big ET-1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET-1 with amounts in the range 120-160 pmol.6. The hydrolysis profile of ET-1 by E-24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.7. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E-24.11 and could be useful for the design of ECE inhibitors. Since E-24.11 can both synthesize and hydrolyse ET-1, the presence of E-24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.

Rapid degradation of endothelin-1 by an enzyme released by the rat isolated perfused mesentery.

1. In vivo the effects of endothelin-1 (ET-1) are limited by its rapid removal from the circulation and possibly by its metabolism by enzymes such as neutral endopeptidase 24.11, deamidase or carboxypeptidase A. Here, using as a model the isolated perfused mesenteric arterial bed of the rat, we have examined the involvements of these enzymatic activities in the vascular responses to ET-1. 2. Samples of Krebs buffer which had been recirculated through the mesenteric arterial bed for 30 min rapidly destroyed the activity of ET-1 as assessed either by bioassay on rings of rat thoracic aorta or by high performance liquid chromatography (h.p.l.c.). For instance, after 15 min incubation with the recirculated-Krebs solution (recirc-K) the contraction induced by 3 x 10(-9) M ET-1 was reduced by more than 90%. Contractions induced by sarafotoxin 6b (3 x 10(-9) M) were similarly suppressed by preincubation with recirc-K whereas those to Arg-vasopressin (3 x 10(-9) M) were unaffected. 3. The degradation of ET-1 by recirc-K was prevented by 1,10-phenanthroline (10(-3) M), abolished by heating the recirc-K solution to 90 degrees C for 15 min, and reduced by EGTA (5 x 10(-3) M) or ET-1(16-21) (10(-5) M). For instance, in the presence of ET-1(16-21) (n = 6) the contraction induced by ET-1 was reduced by only 40% after 15 min incubation with recirc-K buffer. Leupeptin (3 x 10-4 M), dichloroisocoumarin(5 x 10-5 M), phenylmethyl-sulphonyl fluoride (10-3 M), a combination of bacitracin (300 mg ml-1),bestatin (10-5 M), captopril (10-5 M), phosphoramidon (10-4 M) and thiorphan (10-4 M) or Polypep (aproprietary protein digest) did not inhibit the degradation of ET-1 by recirc-K.4. In experiments examining directly the vascular responses of the isolated perfused mesentery of the rat, the addition of cumulative concentrations of ET-1 to the recirculating Krebs solution caused small concentration-dependent increases in perfusion pressure. The inclusion of ET-1(16-2l), ET-1(17-21), or ET-1(18-21) (10-5M) greatly potentiated these responses, but not those to Arg-vasopressin or methoxamine.The effects of 1,10-phenanthroline or EGTA could not be examined in this system because these agents both depressed non-specifically the vasoconstrictor responses of the mesenteric vascular bed.5. Thus, the rat mesentery releases an enzyme that very rapidly destroys ET-1 or the very closely related peptide, sarafotoxin 6b but not Arg-vasopressin. This enzyme is most probably a metallopeptidase because of its sensitivity to inhibition by 1,10-phenanthroline or EGTA. It is particularly interesting that a simple vascular bed such as the mesentery produces such a powerful endothelin metabolising enzyme. It is tempting, therefore, to speculate that the endothelin degrading enzyme active at neutral pH that- we have found is important in the metabolism of ET-1 throughout the vasculature.

Evidence from receptor antagonists of an important role for ETB receptor-mediated vasoconstrictor effects of endothelin-1 in the rat kidney.

1. To characterize the receptor subtype(s) mediating the renal vasoconstrictor effects of the endothelin (ET) and sarafotoxin (SX) peptides in the isolated perfused kidney of the rat, we have examined the effects of endothelin-1 (ET-1), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists, BQ-123 and FR 139317 as selective ETA receptor antagonists, and PD 145065 as a non-selective (ETA and ETB) receptor antagonist. We have also compared in the anaesthetized rat the systemic pressor and renal vasoconstrictor effects of ET-1 and SX6c alone or after pretreatment with PD 145065. 2. In the isolated perfused kidney, ET-1, SX6b and SX6c all gave similar concentration-dependent increases in perfusion pressure. The ETA receptor selective antagonists, BQ-123 and FR 139317, both partially blocked the increase in perfusion pressure induced by ET-1. In contrast, PD 145065 completely blocked the increase in perfusion pressure caused by ET-1. 3. Indomethacin (10 microM) had no effect on the ET-1-induced increases in perfusion pressure but significantly reduced the vasoconstriction induced by low concentrations of SX6c, without affecting responses to high concentrations. In the anaesthetized rat, indomethacin (5 mg kg-1) did not modify the systemic pressor or renal vasoconstrictor effects of ET-1 or SX6c. 4. In anaesthetized rats, bolus intravenous injections of ET-1 or SX6c (0.1, 0.25, 0.5 or 1.0 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP). Both peptides caused an equipotent fall in renal blood flow (RBF).PD 145065 (5 mg kg-1) partially antagonized the systemic pressor effects of ET-1 and SX6c but completely blocked the fall in RBF and rise in renal vascular resistance (RVR) induced by ET-1 and SX6c. PD 145065 also antagonized the transient depressor effect following the bolus administration of either ET-1 or SX6c.5. These results indicate that ET/SX induced renal vasoconstriction is mediated via ETA and ETB-like receptors with ETB receptors having a predominant role in vivo. This may be of therapeutic relevance for an ETA receptor-selective antagonist may offer only limited protection against the deleterious renal effects of endogenous ETs.

Contractile activity of endothelin precursors in the isolated gallbladder of the guinea-pig: presence of an endothelin-converting enzyme.

A single addition of 3 x I0-7 M ET-1, ET-2 or ET-3 produced contractions that reached a steady state in 28.2 +/- 4.2, 21.1 +/- 1.3 and 24.0 +/- 3.8 min, respectively and took 2.7 +/- 0.4, 2.1 + 0.1 and 1.6 +/- 0.1 min to reach half of this steady-state response.4. Contractions induced by 3 x I0-7 M big ET-11-38 or big ET-11- 39 reached a plateau in 38.5 +/- 3.6 and 35.6 +/- 3.3 min, respectively, and half of these responses were attained in 12.0 +/- 2.5 and 7.1 +/- 1.1 min.Thus, these contractions developed more slowly than those induced by ET-1. Contractions induced by 3 x 10-7 M big ET-21-38 were also much slower to develop than those to ET-2, for these took 49 +/- 2 min to reach plateau and 19.4 +/- 2.1 min to attain half that response. Contractions induced by 3 x 10-7 M big ET-31-41 amide took 50.2 +/- 3.7 min to reach a plateau and 27.3 +/- 3.0 min to reach half of this response.5. Phosphoramidon (0.1, 1 and 3 x 10-4 M) inhibited contractions induced by big ET-11.39. For instance,the contractions induced by 3 x 10-7 M big ET-11-39 were inhibited by 10-4 M or 3 x 10-4 M of phosphoramidon by 62.8 +/- 6.7% or 74.5 +/- 4.6%, respectively. Similarly, contractions induced by ET-21-38 were inhibited by 91.3 +/- 5.4% and the small response induced by big ET-3l-4l amide was abolished by 3 x 10-4M phosphoramidon. Conversely, the neutral endopeptidase (EC 24.11) inhibitor DL-thiorphan(3 x 10-4 M) had no effect. Captopril (10-5 M), pepstatin A (10-5 M), phenylmethylsulphonylfluoride(PMSF, 10-3 M), aprotinin (10-5 M), E-64 (10-5 M), cystatin (10-6 M), leupeptin (10-4 M),chymostatin (10-4 M), or bestatin (10-5 M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 x 10-9 M big ET-11-39. Only captopril (10-5 M) or leupeptin(10-4 M) increased the contraction induced by 3 x 10-7 M big ET-11-39. Phosphoramidon (10-4 M),pepstatin (10-5 M) or PMSF (10-3 M) did not affect contractions induced by ET-1.6. Removal of the epithelium increased by 70% the size of the contraction induced by 5 microM histamine(1.08 +/- 0.05 g; n = 160 to 1.84 +/- 0.14 g; n = 12) but did not affect, in absolute terms, the contraction induced by ET-1 (as a % of the response to histamine, these responses were, of course, apparently depressed). Epithelium removal did, however, increase the size of the contractions induced by 3 to 30 x 10-9 M big ET-1 -39 which was very similar to the effect of the protease inhibitors.7. In competition binding studies on membranes prepared from the guinea-pig gallbladder, 10-11 MET-1 inhibited by 76.9 +/- 3.1% the binding of [125]-ET-I while porcine big ET-11-39 caused no inhibition(0.7 +/- 3.0; n = 3). ET-1 (10-6 M) inhibited binding by 95.7 =/- 1.1% (n = 3) while at this much higher concentration, big ET-11-39 inhibited binding by only 16.8 +/- 4.2% (n = 3). This clearly suggests that big ET-11-39 does not bind directly to ET receptors.8. Thus, a phosphoramidon-sensitive endothelin-converting enzyme (ECE), different from neutral endopeptidase (NEP; EC 24.11) and not located on the epithelium, converts big ET-1 into ET-1 in the gallbladder of the guinea-pig. This ECE appears to act preferentially on big ET-1 or big ET-2 over bigET-3.

Effects of glucocorticoid treatment and acute passive immunization with growth hormone-releasing hormone and somatostatin antibodies on endogenous and stimulated growth hormone secretion in the male rat.

Despite causing marked inhibition of somatic growth, glucocorticoids enhance both the response to GH-releasing hormone (GHRH) and the amplitude of naturally occurring GH secretory pulses in the male rat. The relative contribution of the two major hypothalamic regulatory factors for GH (somatostatin and GHRH) to these observed effects remains speculative. In the present studies, we have investigated endogenous and stimulated GH release in rats pretreated with glucocorticoid or vehicle, and the effects of passive immunoneutralization of somatostatin or GHRH. In an initial study, four groups of eight rats were treated with either saline or various doses of a depot preparation of betamethasone: low dose, 0.85 mg; medium dose, 1.7 mg; high dose, 3.4 mg. All doses significantly suppressed body weight gain, total adrenal weight and concentrations of both plasma corticosterone and pituitary ACTH. Seven days after betamethasone treatment, GH responses to an i.v. injection of 1 microgram human GHRH(1-29) were evaluated during pentobarbitone anaesthesia. Compared with saline-treated controls (peak GH concentration of 506.0 +/- 68.5 micrograms/l), peak GH levels were enhanced by the low dose (704.4 +/- 47.8 micrograms/l, P less than 0.05), unaltered by the medium dose (543 +/- 65.8 micrograms/l) and suppressed by the high dose (312.7 +/- 55.2 micrograms/l, P less than 0.05) of betamethasone. Similarly, the area under the secretory curves was increased by 46% following the low dose (P less than 0.01), unaltered by the medium dose and reduced by 33% after the high dose of betamethasone. In a second study, rats were pretreated for 7 days before blood sampling with either the medium dose of betamethasone or saline. On day 5, 48 h before blood sampling, an indwelling venous catheter was fitted enabling sampling of conscious rats. On the day of study, blood samples were taken at 30-min intervals over an initial 2-h period (10.00-12.00 h). Following the sample at 12.00 h, rats were given the reconstituted and dialysed immunoglobulin fraction from either control sheep serum (NSIgG), sheep anti-rat GHRH serum (GHRHab) or sheep anti-somatostatin serum (SRIHab), and samples were taken for a further 90 min (12.30-14.00 h). Directly after the sample at 14.00 h, GH stimulation was effected in all rats using 1 microgram human GHRH(1-29) with samples taken at 5, 10, 20 and 40 min following stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)