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There is evidence in animals and in humans for accelerated natriuresis after oral compared with intravenous sodium loading. To assess the role of atrial natriuretic peptide (ANP) as a contributory mechanism, we compared the hormonal responses to an intravenous sodium load and to the same sodium load taken orally in three separate groups of healthy subjects in balance on low, normal, or high sodium intake. On each diet, there was a trend for an early delay in sodium excretion, followed by increased natriuresis after the oral compared with intravenous sodium load. On all levels of dietary sodium intake, there was a significant (∼2-fold) increase in plasma ANP levels after intravenous saline infusion. There was a significant suppression of the renin system both after oral and intravenous sodium loading. However, there was no acute increase in plasma ANP levels after the oral sodium load, except on the very low sodium intake. This striking and unexpected observation suggests that changes in plasma ANP levels appear to play little role in the early response to an acute oral sodium load in subjects with sodium intake in the range of 150–350 mmol/day. Endocrine mechanisms for the accelerated increase in sodium excretion after oral compared with intravenous sodium loading remain to be elucidated.

1. Although mast cell hyperplasia is a feature of rheumatoid arthritis and osteoarthritis, the extent and nature of mast cell activation in joint disease have not been clearly established. 2. We have investigated the levels of mast cell tryptase and histamine and also of eosinophil cationic protein in synovial fluid collected from 31 patients with rheumatoid arthritis, 14 with seronegative spondyloarthritis and nine with osteoarthritis. Two RIAs for tryptase were employed: one with monoclonal antibody AA5, which was found to bind equally well to both α and β isoforms on Western blots of the recombinant enzyme, and the other with antibody G5, which recognizes predominantly β-tryptase. 3. α-Tryptase, which is likely to be released constitutively from mast cells, appeared to be the major form in synovial fluid, as the assay with antibody AA5 detected appreciably more tryptase than that with antibody G5. β-Tryptase, which is released on anaphylactic activation of mast cells, was detected in 14 out of 45 synovial fluid samples studied, with concentrations of up to 12 μg/l measured by the G5 assay. The apparent levels of β-tryptase, but not of α-tryptase, were closely correlated with those of histamine in the synovial fluid. Patients with osteoarthritis appeared to have a greater proportion of β-tryptase in the synovial fluid than those with rheumatoid arthritis, as well as higher concentrations of histamine. Eosinophil cationic protein was present at high levels in the synovial fluid, although eosinophil numbers were low, and its concentrations were not correlated with the concentrations of the mast cell products. 4. These data suggest that anaphylactic degranulation of mast cells may have occurred to a greater extent in osteoarthritis than in rheumatoid arthritis, despite the relative lack of synovial inflammation in osteoarthritis. Although the eosinophil cationic protein detected may not reflect eosinophilic inflammation in the joint, the presence in synovial fluid of tryptase of both major forms, and of histamine, appears to indicate that mast cell products are secreted constitutively, as well as by processes of anaphylactic degranulation in rheumatoid arthritis, seronegative spondyloarthritis and osteoarthritis.

Hormonal and renal effects of candoxatril, a neutral endopeptidase 24.11 inhibitor, were investigated in eight subjects equilibrated on a low sodium diet (10 mmol sodium per day) and a high sodium (350 mmol per day) diet. After candoxatril treatment, plasma ANP increased to a maximum at 2-4 h and declined to baseline within 24 h. The increases were relatively greater on the high sodium diet, which was also associated with increases in urinary sodium, with highest values at 4h. On the low sodium diet, the magnitude of the changes was significantly lower (24 h cumulative sodium excretion was 11.4 +/- 5.5 mmol on the low sodium diet and 73.1 +/- 25.6 mmol on the high sodium diet; P < 0.01). There were no significant effects on urinary potassium excretion, creatinine clearance or haematocrit. After candoxatril treatment there were reductions in PRA, especially on the low sodium diet. On either diet there were no effects on systemic blood pressure. These results demonstrate that dietary sodium intake is an important determinant of the renal and hormonal responses to neutral endopeptidase inhibition.

1. The aim of this study was to determine plasma levels of N-terminal atrial natriuretic peptide and atrial natriuretic peptide in normal subjects and in patients with essential hypertension, cardiac transplant and chronic renal failure, using radioimmunoassays directed towards the mid-portion pro-atrial natriuretic peptide (31-67) and pro-atrial natriuretic peptide (1-30) of the N-terminal atrial natriuretic peptide and atrial natriuretic peptide (99-126). The circulating form(s) of the immunoreactive N-terminal atrial natriuretic peptide in plasma extracts has been investigated using all three radioimmunoassays by means of gel filtration chromatography to further clarify the major immunoreactive molecular circulating form(s) of N-terminal atrial natriuretic peptide in man. 2. The plasma level (mean ± SEM) of N-terminal pro-atrial natriuretic peptide (31-67) in the normal subjects was 547.2 ± 32.7 pg/ml (n = 36) and was significantly elevated in patients with essential hypertension (730.2 ± 72.3 pg/ml, P < 0.025, n = 39), in cardiac transplant recipients (3214.0 ± 432.2 pg/ml, P < 0.001, n = 9) and in patients with chronic renal failure (3571.8 ± 474.1 pg/ml, P < 0.001, n = 11). Plasma levels of N-terminal pro-atrial natriuretic peptide (1-30) and atrial natriuretic peptide were similarly elevated in the same patient groups when compared with the mean plasma values in the normal subjects. 3. There were positive associations between pro-atrial natriuretic peptide (31-67) and atrial natriuretic peptide, pro-atrial natriuretic peptide (31-67) and pro-atrial natriuretic peptide (1-30) and between pro-atrial natriuretic peptide (1-30) and atrial natriuretic peptide in the normal subjects, hypertensive patients, cardiac transplant recipients and patients with chronic renal failure. The correlation coefficient for all groups taken together was 0.86 (P < 0.001. n = 95) for pro-atrial natriuretic peptide (31-67) and atrial natriuretic peptide, 0.93 (P < 0.001, n = 95) for pro-atrial natriuretic peptide (31-67) and pro-atrial natriuretic peptide (1-30), and 0.82 (p < 0.001, n = 95) for pro-atrial natriuretic peptide (1-30) and atrial natriuretic peptide. 4. Gel filtration of extracted plasma from cardiac transplant patients and patients with chronic renal failure indicated a single peak of immunoreactivity for N-terminal atrial natriuretic peptide using both the pro-atrial natriuretic peptide (31-67) and pro-atrial natriuretic peptide (1-30) radioimmunoassays, suggesting a major single high-molecular-mass circulating immunoreactive N-terminal atrial natriuretic peptide, probably pro-atrial natriuretic peptide (1-98). Atrial natriuretic peptide immunoreactivity, as measured by the radioimmunoassay for atrial natriuretic peptide (99-126), showed a separate and distinct peak from that of the N-terminal atrial natriuretic peptide, which co-eluted with the synthetic human standard atrial natriuretic peptide (99-126). 5. These results show that immunoreactive N-terminal atrial natriuretic peptide and atrial natriuretic peptide are elevated in patients with essential hypertension, in cardiac transplant recipients and in patients with chronic renal failure. The major immunoreactive form of N-terminal atrial natriuretic peptide cross-reacting in both the pro-atrial natriuretic peptide (31-67) and pro-atrial natriuretic peptide (1-30) radioimmunoassays is of a high molecular mass, probably pro-atrial natriuretic peptide (1-98). Since pro-atrial natriuretic peptide (1-98) is unlikely to cross-react identically with antibodies for pro-atrial natriuretic peptide (31-67) or pro-atrial natriuretic peptide (1-30), this could account for the differences in plasma levels obtained by the assays for pro-atrial natriuretic peptide (31-67) and pro-atrial natriuretic peptide (1-30).

1. We have developed a radioimmunoassay for the measurement of immunoreactive brain natriuretic peptide (1–32) in human plasma. Simultaneous measurements of atrial natriuretic peptide have also been carried out to allow for direct comparison between circulating brain natriuretic peptide and atrial natriuretic peptide. Plasma levels of immunoreactive brain natriuretic peptide (means ± sem) were 1.1 ± 0.1 pmol/l in 36 normal healthy subjects and were significantly elevated in cardiac transplant recipients (18.8 ± 3.9 pmol/l, n = 12) and in patients with dialysis-independent (8.8 ± 1.5 pmol/l, n = 11) or dialysis-dependent (41.6 ± 8.8 pmol/l, n = 14) chronic renal failure. Similarly, in these groups of patients plasma levels of atrial natriuretic peptide were also significantly raised when compared with those in the group of normal healthy subjects. 2. The plasma level of atrial natriuretic peptide was significantly higher than that of brain natriuretic peptide in normal subjects and in patients with dialysis-independent chronic renal failure, with ratios (atrial natriuretic peptide/brain natriuretic peptide) of 2.8 ± 0.2 and 2.2 ± 0.3, respectively. However, in both cardiac transplant recipients and patients on dialysis plasma levels of atrial natriuretic peptide and brain natriuretic peptide were similar, with ratios of 1.3 ± 0.2 and 1.0 ± 0.1, respectively, in these two groups. 3. Plasma levels of brain natriuretic peptide and atrial natriuretic peptide were significantly correlated in the healthy subjects and within each group of patients. When all groups were taken together, there was an overall correlation of 0.90 (P 4. Patients on dialysis had the highest plasma levels of both brain natriuretic peptide (41.6 ± 8.8 pmol/l, n = 14) and atrial natriuretic peptide (41.3 ± 9.4 pmol/l, n = 14) and the levels of both peptides declined significantly after maintenance haemodialysis. However, the overall percentage decrease in the plasma level of atrial natriuretic peptide (43.6 ± 7.5%) after dialysis was significantly greater than that observed for brain natriuretic peptide (15.9 ± 5.3%, P 5. Displacement curves of iodinated atrial natriuretic peptide from bovine adrenal membranes by human atrial natriuretic peptide (99–126) and human brain natriuretic peptide (1–32) gave a median inhibitory concentration of 144 pmol/l for atrial natriuretic peptide and 724.4 pmol/l for brain natriuretic peptide. The cross-reactivity of human brain natriuretic peptide with the atrial natriuretic peptide receptor preparation was 19.5% of that of atrial natriuretic peptide, indicating that human brain natriuretic peptide has a lower binding affinity for the atrial natriuretic peptide receptor/binding site on bovine adrenal membranes. 6. These results suggest that brain natriuretic peptide is co-secreted with atrial natriuretic peptide and may also be an important factor in the adaptive mechanisms to impairment of renal function. However, whether brain natriuretic peptide has an independent and fundamentally important role in man remains to be investigated.

Basal atrial natriuretic peptide levels and the response to exogenous atrial natriuretic peptide are influenced by dietary sodium intake. In view of interest in the therapeutic potential of elevating plasma atrial natriuretic peptide by inhibition of neutral endopeptidase 24.11, we studied the renal and hormonal effects of 200 mg of the oral endopeptidase 24.11 inhibitor candoxatril in eight patients with untreated essential hypertension on high sodium (350 mmol/day) and low sodium (10 mmol/day) diets. With endopeptidase 24.11 inhibition, plasma atrial natriuretic peptide increased more than twofold on low and high sodium diets (p less than 0.05). Plasma N-terminal pro-atrial natriuretic peptide increased on the high sodium intake but was unaffected by candoxatril. Urinary sodium excretion increased threefold on the low sodium and sixfold on the high sodium diet (p less than 0.05). The absolute increase in urinary sodium excretion during the 24 hours after treatment compared with placebo was 18 +/- 8 mmol on the low sodium and 98 +/- 34 mmol on the high sodium diet (p less than 0.05). Plasma renin activity was suppressed by treatment on the low but not on the high sodium diet (p less than 0.05). Blood pressure did not change in the 6 hours after a single dose of candoxatril. These findings show that sodium intake is a major determinant of the response to endopeptidase 24.11 inhibition. The lack of effect on N-terminal pro-atrial natriuretic peptide suggests that candoxatril does not influence cardiac secretion of atrial natriuretic peptide or catabolism of N-terminal pro-atrial natriuretic peptide, and the latter does not appear to play a role in the response to candoxatril.

1. The possible natriuretic and kaliuretic effects of a single dose of lithium, as used in lithium clearance studies, were investigated in 15 healthy subjects on fixed sodium (100 mmol/24 h) and potassium (70 mmol/24 h) intakes. Lithium carbonate (300 mg or 600 mg) or placebo tablets were administered, double-blind and in random order, midway through a 48 h urine collection (divided into six 8 h periods), at 23.00 hours. 2. During the three 24 h periods which preceded the administration of lithium or placebo (control days), rates of sodium and potassium excretion followed normal circadian patterns, but no differences in excretion rates between the 3 control days were observed. Placebo tablets did not affect excretion rates. 3. After the 300 mg dose of lithium carbonate, 24 h sodium excretion increased by approximately 17 mmol (P < 0.05); almost all of the natriuretic effect occurred during the first two 8 h periods. No effect on potassium excretion was observed. 4. After the 600 mg dose of lithium carbonate, 24 h sodium excretion increased by approximately 48 mmol (P < 0.001) and 24 h potassium excretion increased by approximately 19 mmol (P < 0.01). These effects were confined to the first two 8 h periods and thus occurred before and during the usual lithium clearance period. 5. Plasma renin activity, measured in 10 subjects, increased after the 600 mg dose of lithium carbonate (P < 0.005), but plasma concentrations of aldosterone and atrial natriuretic peptide were not significantly affected. Neither the 300 mg dose of lithium carbonate nor the placebo tablets affected hormone levels. 6. It is recommended that the test dose of lithium carbonate for use in lithium clearance studies should not exceed 300 mg.

Twelve patients with essential hypertension who were already on treatment with the long-acting calcium antagonist amlodipine (5 mg once daily) were entered into a double-blind, randomized crossover study of the addition of one month's treatment with either bendrofluazide (5 mg once daily) or matching placebo. The addition of bendrofluazide did not cause any statistically significant fall in the supine blood pressure compared to treatment with placebo (147.6/90.1 +/- 4.8/2.8 v 150.8/92.6 +/- 4.3/2.3 mm Hg, respectively). Plasma potassium was significantly lower on bendrofluazide as compared to placebo (3.11 +/- 0.14 v 3.62v +/- 0.13 mmol/L, P less than .001) and 10 of 12 patients had a fall in plasma potassium while on diuretic. The results of this study suggest that a thiazide diuretic has little additive effect on blood pressure in patients already on the long-acting dihydropyridine amlodipine, and may cause hypokalemia.

OBJECTIVE--To assess the changes in sodium excretion and sodium balance after withdrawal of long term nifedipine. DESIGN--Single blind, placebo controlled study in patients receiving fixed sodium and potassium intakes. SETTING--Blood pressure unit of a teaching hospital in south London. PATIENTS--Eight patients with mild to moderate uncomplicated essential hypertension who had been taking nifedipine 20 mg twice daily for at least six weeks. INTERVENTIONS--Withdrawal of nifedipine and replacement with matching placebo for one week. MAIN OUTCOME MEASURES--Urinary sodium excretion and cumulative sodium balance, body weight, plasma atrial natriuretic peptide concentrations, plasma renin activity and aldosterone concentrations, and blood pressure. RESULTS--During nifedipine withdrawal there was a significant reduction in urinary sodium excretion (day 1: -62.7 mmol/24 h; 95% confidence interval -90.3 to -35.0) and each patient retained a mean of 146 (SEM 26) mmol sodium over the week of replacement with placebo. Body weight and plasma atrial natriuretic peptide concentrations increased during the placebo period and seemed to be associated with the amount of sodium retained. Systolic blood pressure rose from 157 (9) to 165 (9) mmHg (95% confidence interval of difference -7.1 to 22.1) when nifedipine was replaced with matching placebo, and the rise seemed to be related to the amount of sodium that was retained. CONCLUSIONS--Nifedipine causes a long term reduction in sodium balance in patients with essential hypertension. This long term effect may contribute to the mechanism whereby nifedipine lowers blood pressure.

The effects of alterations in dietary potassium (40, 80 and 160 mmol day-1) on endocrine status and on renal lithium clearance were assessed in 10 healthy subjects on a fixed sodium intake; measurements were made on the fifth day of each dietary regimen. Plasma aldosterone concentration was found to increase with potassium intake, whereas plasma renin activity and the plasma concentration of atrial natriuretic peptide did not change significantly. Neither absolute lithium clearance nor fractional lithium excretion was affected measurably by changes in dietary potassium, suggesting that provided the potassium intake remains within the normal range it is unnecessary to control this factor during lithium clearance studies in man.

Matrix metalloproteinases (MMPs) are involved in a number of physiological and pathologic processes including the inflammation found in IBD. We have shown that MMP-3 is upregulated in Crohn's disease and in ulcerative colitis. This study shows a potential role for MMP-12 in these idiopathic diseases.

Sudden Infant Death Syndrome, (SIDS) or cot death, remains the most common category of post-perinatal death in the UK. By definition, the cause of death is unknown, but a long-standing theory is that some of these deaths could be the result of anaphylaxis.To investigate the potential contribution of anaphylactic mechanisms to deaths in infancy by determining relative levels of alpha- and beta-tryptases and both total and allergen-specific IgE in sera from groups of infants whose deaths were attributed to SIDS or to other causes.Serum samples were collected at the time of post-mortem examination from infants whose death was classed as SIDS (n = 40) and from a comparison group in which cause of death had been established (n = 32). Serum tryptase concentrations were measured with a radioimmunoassay with monoclonal antibody G5 which detects primarily beta-tryptase or an ELISA with antibody AA5 which has equal sensitivity for alpha- and beta-tryptases. Levels of total IgE and IgE specific for casein, beta-lactoglobulin, house dust mite and moulds were determined.Analysis of the results of the two assays for tryptase indicated that levels of the beta-like tryptase (the form secreted on anaphylactic degranulation) were significantly higher in serum from infants with SIDS compared with those whose death was explained. There was no evidence for an increase in serum levels of alpha-tryptase (the variant secreted constitutively from mast cells). Total levels of serum IgE did not differ between the two groups and, reflecting the low circulating IgE concentrations in infancy, an elevation in IgE specific for the panel of allergens was not detected.In a proportion of SIDS victims there may be increased serum levels of beta-like tryptase, a marker for anaphylaxis. The failure to detect an increase in alpha-tryptase would suggest that mast cell hyperplasia is not a feature of cot death. The nature of the inciting agents remains unclear, but anaphylaxis deserves serious consideration as a possible cause of sudden death in infancy.

C-type natriuretic peptide (CNP) is a potent, endothelial-derived relaxant and growth-inhibitory factor. Accelerated vascular disease is an important cause of morbidity in cardiac transplant recipients, and endothelial dysfunction is now well recognized in patients with cardiovascular disease. CNP has not previously been investigated following cardiac transplantation. We therefore studied plasma levels of immunoreactive CNP in patients early and late after heart transplantation, compared with levels in healthy subjects. We measured CNP in extracted human plasma using an antibody against human CNP-(1-22). CNP levels were significantly elevated in 13 cardiac recipients 2 weeks post-transplant [2.64+/-0.26 pmol/l (mean+/-S.E.M.)] compared with those in the normal healthy subjects (0.62+/-0.04 pmol/l; n=20, P0.001). Plasma levels of CNP were also significantly elevated in a second group of established cardiac transplant recipients (1.15+/-0.07 pmol/l; n=46) studied 1-13 years post-transplant when compared with the healthy subjects (P0.001). In the group studied later after transplantation, CNP levels were significantly associated with systolic blood pressure (P0.05) and were higher in patients with angiographic post-transplant coronary artery disease (P=0.032). In conclusion, these findings clearly demonstrate that CNP is elevated soon after cardiac transplantation and remains raised in patients even several years post-transplant. CNP may be important as a circulating or local hormone involved in vascular contractile function and in the pathophysiology of cardiac allograft vasculopathy following heart transplantation.

Chymase is an important marker for human mast cells as well as a mediator of inflammation and matrix remodelling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. A monoclonal antibody to chymase (designated CC1) was prepared by immunizing a mouse with chymase purified from human skin, fusing the splenocytes with NS-1 myeloma cells, and screening the hybridoma supernatants by ELISA with recombinant human prochymase isolated from a baculovirus expression system. This antibody bound to chymase in western blots and bound selectively to cells with the morphology and distribution of mast cells in paraffin wax sections of skin, synovium, lung, and heart. In sequential sections and with double-labelling experiments, chymase was localized to cells which contained mast cell tryptase; in contrast to previous reports, no evidence was found for its presence in endothelial cells or any other cell type. The antibody permitted chymase-containing mast cells to be detected in formalin-fixed tissues, and the numbers identified were similar to those in tissues fixed with Carnoy's or ethanol fixatives. Immunocytochemistry with antibody CC1 provides for the first time a sensitive and specific means for the detection of chymase in routinely fixed tissues and should prove valuable in studying mast cell subsets in disease.

Despite increasing evidence that basophils can infiltrate into inflamed tissues during allergic reactions, determination of the extent of infiltration and elucidation of their role in allergic disease has been frustrated by the lack of reliable means for detecting this cell type in tissues. In the present study, we report on a new monoclonal antibody specific for basophils and on the initial characterization of the antigen it recognizes. Basophils were isolated from peripheral blood by Percoll density gradient centrifugation and a positive-selection immunomagnetic procedure and injected into mice to produce monoclonal antibodies. A hybridoma clone, designated BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively to basophils on immunocytochemistry but did not react with any other cell type or tissue structure, although it did stain a proportion of cells from the basophilic cell line KU812F. In sections of mixed populations of peripheral blood cells, similar numbers of cells stained with Alcian blue dye and BB1 over a wide range of basophil purity. BB1 antibody was effective in identifying basophils in sections of mixed cells or in tissues after fixation with ethanol, Carnoy's solution, or formalin. Staining of basophils with BB1 gave a granular appearance, although flow cytometry indicated that some antigen was also present on the surface of the cell. Activation of these cells with anti-IgE antibody or with the calcium ionophore A23187 provoked release of the antigen in parallel with that of histamine. BB1 antibody did not, by itself, stimulate histamine release. The molecular mass of the antigen was determined on Hedrick-Smith gels to be 124+/-11 kd. This new monoclonal antibody will be a valuable experimental tool in future studies, allowing the reliable detection of basophils in tissues of patients with allergic and chronic inflammatory disease; in addition, the antigen it identifies has potential as a unique marker of basophil activation.

Although there is relatively little evidence of inflammation in osteoarthritis (OA), increases in mast cell numbers and mast cell activation are prominent features of the synovial tissue. As little is known of the types of mast cells which may be involved, the numbers and distribution of mast cell subpopulations have been investigated as defined according to their content of proteases. Tissue was obtained from patients with OA undergoing total knee replacement surgery (n = 14) and from control subjects either post-mortem (n = 11) or following leg amputation for peripheral vascular disease (n = 3); a double-labelling immunocytochemical procedure with monoclonal antibodies specific for tryptase and chymase was applied to identify those mast cells which contain both tryptase and chymase (MCTC) and those with tryptase but not chymase (MCT). There was considerable variation between individual tissues and between sites of tissue sampling, but cells of the MCTC subset were predominant in the synovial layer of both groups of subjects without joint disease, accounting for some 60 per cent of all mast cells present. In tissue from OA patients, however, there appeared to have been a striking shift in the relative proportions of mast cells from the MCTC to the MCT phenotype, with many more MCT cells present in the synovial tissues of OA patients (median 53 MCT/mm2) than in tissue from post-mortem (7.5 MCT/mm2, P0.0001) or amputation controls (12 MCT/mm2). In contrast, numbers of synovial MCTC cells in the synovium of OA patients (20 MCTC/mm2) differed little from those in either of the control groups (both 12 MCTC/mm2). In several other conditions, the MCT cells have been linked with inflammatory events, but it seems that in OA, other factors may be operating to induce a selective expansion of this subpopulation.

1. Hormones involved in cardiovascular regulation are influenced by drug treatment. It is therefore difficult to study endocrine mechanisms in heart failure as most patients are already on treatment by the time they reach hospital. 2. We studied nine hospital in-patients before and after treatment of acute New York Heart Association class IV heart failure. 3. Before treatment, plasma brain and atrial natriuretic peptides were markedly elevated (BNP 121 ± 26 pg/ml, ANP 163 ± 33 pg/ml; normal range: BNP 3.9 ± 0.3 pg/ml, ANP 8.6 ± 0.8 pg/ml) and correlated positively with serum creatinine and left ventricular end-diastolic diameter and negatively with ejection fraction. Eight patients improved and one died. 4. With improvement plasma ANP and BNP fell. Initial renin activity was within the normal range but increased on treatment. Plasma neuropeptide Y and adrenaline remained normal before and after treatment in the eight patients who improved. Initial plasma noradrenaline was in the normal range in four of these patients and just above normal in a further four. In the patient who died, initial plasma neuropeptide Y and catecholamines were very high. 5. Plasma BNP emerged as complementary to ANP as a dynamic index in severe heart failure; however, renal function is also an important determinant of plasma BNP and ANP. There is little evidence for activation of the circulating renin—angiotensin—aldosterone system or neuropeptide Y before treatment of acute heart failure.

1.BNP and ANP are important research indices of severity of heart failure. However, uncertainty regarding the stability of these peptides at room temperature has limited their use to assess cardiac function in routine clinical practice. 2. We assessed the stability of BNP and ANP in blood samples left for 2 h or 2 days at room temperature compared with levels in blood processed immediately (initial). These times were chosen to reflect possible times for samples to be processed in a hospital outpatient clinic (2 h) or a blood sample posted to a laboratory from general practice (2 days). Samples were obtained from eight heart transplant recipients. Blood was separated and plasma stored immediately after collection (initial) and after 2 h or 2 days at room temperature respectively. 3. Initial plasma BNP and ANP values measured by radioimmunoassay after Sep-Pak extraction were 38.9+/-11.1(S.E.M.) pg/ml and 113.6+/-28.1 pg/ml, respectively. After 2 h at room temperature there was no significant fall in either peptide level (35.5+/-9.9 pg/ml, BNP; 104. 9+/-30.6 pg/ml, ANP). However, after 2 days at room temperature there was a significant fall in ANP to 38.1+/-12.6 pg/ml (P0.005 versus initial level). In contrast, there was no significant fall in BNP after 2 days (32.0+/-8.4 pg/ml). After 2 days at room temperature only 30.4+/-4.3% of the ANP remained, but 86.0+/-5.0% of BNP compared with the initial ANP and BNP measurements. 4. Our study clearly showed that ANP is stable for 2 h and thus could be useful as a screening test for heart disease in hospital. In contrast, BNP remained stable for 2 days. Measuring BNP may thus be practical as a test of heart function both for routine use in hospital and by general practitioners in the community.

This study has examined the association between circulating atrial natriuretic peptide (ANP), plasma cyclic GMP and urinary cyclic GMP in relation to hypertension and reduced renal function in 30 normotensives, in 30 patients with essential hypertension and in 22 patients with stable dialysis-independent chronic renal failure (CRF). Plasma ANP was significantly raised (about two-three-fold) in the CRF group compared with the hypertensive and normal groups; plasma cyclic GMP was also significantly raised in the CRF group (median group values: 4.6, 5.8 and 11.0 pmol/ml, respectively, for the normal, hypertensive and CRF groups). There were no significant differences in urinary cyclic GMP between the normotensives and hypertensives but urinary cyclic GMP was significantly reduced in the patients with CRF (median group values: 407.1, 450.9 and 247.8 pmol/min for the normal, hypertensive and CRF groups, respectively, P0.001). In the subjects with CRF, the clearance of cyclic GMP was reduced in proportion to the clearance of creatinine, but there was no significant difference in the fractional excretion of cyclic GMP (median group values: 78.1% in the normal group, 78.9% in the hypertensive group and 70.2% in the CRF group). In all groups, there was no association between circulating ANP and urinary cyclic GMP: By contrast, there was a positive association between plasma ANP and plasma cyclic GMP (r = 0.39 P0.001) that was independent of blood pressure or renal function. These results demonstrate that while a substantial amount of urinary cyclic GMP originates from the glomerular filtrate, to some extent, raised plasma ANP also contributes to the circulating levels of cyclic GMP. However, plasma cyclic GMP cannot be taken as a direct substitute for plasma ANP.

There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn's disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn's disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 +/- 23 cells mm-2; gastritis: 413 +/- 139 cells mm-2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn's patients were actually decreased (34 +/- 30 cells mm-2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn's disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract.

We examined the capacity of interleukin (IL)-4 to induce or enhance the expression of certain cytokines in resting and activated cells of the HMC-1 human leukemic mast cell line. The HMC-1 mast cells were cultured with or without recombinant human IL-4 and then activated with the calcium ionophore ionomycin. Stimulation of non-IL-4-treated cells with ionomycin (10 microM) for periods of 30 min to 8 hr induced expression of mRNA encoding IL-3, IL-4 and IL-8 but was without effect on levels of mRNA for tumour necrosis factor (TNF)-alpha or beta-actin. Culture of the cells with IL-4 (100 ng/ml) for 24 hr led to a small increase in resting levels of mRNA for IL-3 and IL-8 but not for IL-4, TNF-alpha or beta-actin. More notably, the IL-4 treatment produced a pronounced elevation of mRNA for IL-3 and IL-8 when the cells were subsequently activated with ionomycin. The IL-4 treatment produced a negligible effect on IL-4 mRNA, and no effect on TNF-alpha or beta-actin mRNA levels in ionomycin-activated cells. Quantitation of cDNA by competitive polymerase chain reaction (PCR) revealed that the IL-4 treatment produced a sixfold increase in ionomycin-induced levels of cellular IL-3 mRNA, a fourfold increase in induced IL-8 mRNA and less than a twofold increase in induced IL-4 mRNA. The IL-4 treatment led to a 15- to 20-fold increase in ionomycin-induced secretion of IL-3 product and a doubling of induced IL-8 product. These effects of IL-4 were not associated with increased mast cell numbers. We conclude that IL-4 alone is a weak activator of IL-3 and IL-8 gene expression in mast cells, but is able to enhance activation signals in stimulated mast cells leading to transcription and secretion of these two cytokines.

To examine the changes in plasma brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and blood pressure in patients with essential hypertension on a low, normal and high sodium intake.Twelve patients with mild-to-moderate essential hypertension were studied. Plasma, urinary and blood pressure measurements were made with the patients on their usual sodium intake, then on the fifth day of a low (10 mmol/day) and on the fifth day of a high (350 mmol/day) sodium intake, the sequence being allocated randomly.Plasma levels of BNP and ANP increased significantly on the high sodium intake compared with when the patients were on their normal diet. The mean blood pressure on the high sodium intake was not significantly different from that with the patients on their normal diet. In contrast, plasma BNP and ANP decreased on the low sodium intake, but were not significantly different compared with when the patients were on their normal diet. However, there was a significant reduction in the mean blood pressure on the low sodium intake compared with when the patients were on their normal diet. Compared with the normal diet, BNP and ANP plasma levels showed similar percentage decreases on the low sodium intake and similar percentage increases on the high sodium intake.These findings suggest that BNP and ANP are released in response to a common stimulus during changes in dietary sodium intake. The changes in plasma BNP and ANP observed with sodium restriction and sodium loading indicate the potential importance of BNP and ANP as a dual peptide system contributing to the maintenance of sodium balance and blood pressure regulation in patients with essential hypertension, during changes in dietary sodium intake.

To study the possible physiological role of atrial natriuretic peptide (ANP) in the regulation of intraocular pressure (IOP) the effects of an increase of endogenous ANP within the physiological range induced by the neutral endopeptidase 24.11 (NEP) inhibitor candoxatril were examined. In a single masked placebo controlled trial, seven patients were studied with normal IOP (six male, one female; average age 50 (range 37-62 years). Intraocular pressure in each eye was measured after 2 weeks of placebo, after 4 weeks of candoxatril 200 mg twice daily, and during the first 3 days of placebo washout. With 4 weeks of candoxatril, endogenous plasma ANP levels increased from 4.2 (SEM 1.5) to 6.0 (1.5) pmol/l (p < 0.04) and there was a significant decrease in mean arterial pressure from 119 (4) to 110 (3) mm Hg (p < 0.02; 12 hours after treatment). There was a significant reduction in IOP after 4 weeks' treatment with candoxatril (right eye 2.1 (0.8) mm Hg, p < 0.05 paired t test, left eye 2.8 (0.8) mm Hg, p < 0.02). The mean fall in IOP was 11% (4%) in the right eye and 16% (3%) in the left eye and the fall in IOP was greater the higher the initial IOP. The reduction in IOP with chronic NEP inhibition was positively correlated with the increase in ANP levels but not with changes in blood pressure. These findings suggest that ANP may play a physiological role in the regulation of IOP. As the fall in IOP was greater in subjects with higher initial IOP, NEP inhibitors may be of therapeutic value in the management of glaucoma.

BACKGROUND The role of cardiac extrinsic innervation in the regulation of sodium balance and blood pressure is controversial. METHODS AND RESULTS We performed a double-blind study of endocrine and blood pressure responses to 5 days of low- (LS, 10 mmol/d) and 5 days of high- (350 mmol/d) sodium intake in 12 cardiac transplant recipients, 12 matched healthy subjects, and 12 matched subjects with untreated essential hypertension. In transplant recipients on low sodium, supine blood pressure was 137/94 +/- 8/4 (mean +/- SEM) mm Hg and plasma atrial natriuretic peptide (ANP) was 59.3 +/- 6.3 pg/mL; on high sodium, blood pressure was 148/97 +/- 5/3 mmHg (P < .05 for systolic pressure versus LS), and ANP was 94.3 +/- 10.6 pg/mL (P < .01 versus LS), respectively. Plasma ANP for those on each diet was significantly higher in the cardiac transplant recipients than in healthy or hypertensive controls; relative changes in plasma ANP in changing from low- to high-sodium diet were similar in each group. Urinary sodium excretion by the fifth day of each diet was similar in each group. Suppression of plasma renin activity and aldosterone by high-sodium diet was blunted in cardiac transplant recipients compared with healthy subjects (respectively, plasma renin activity: 1.41 +/- 0.30 versus 0.68 +/- 0.21 ng.mL-1 x h-1, P < .05; aldosterone: 391 +/- 35 versus 166 +/- 21 pmol/L, P < .05). CONCLUSIONS These results suggest that extensive denervation of the heart does not result in major abnormalities in regulation of large changes in sodium intake and that intact cardiac innervation is not required for plasma ANP responses to altered sodium intake. Blood pressure after cardiac transplantation is sensitive to reduced sodium intake.

The objective of this study was to investigate the relationships between erythrocyte Na-Li countertransport, plasma renin activity (PRA), plasma aldosterone and plasma atrial natriuretic peptide (ANP) in a population-based study, in a cross-sectional observation of a randomly selected sample of a male working population, conducted at the Medical Care Center at the Olivetti Factory in Pozzuoli (Naples). One hundred and seventy-nine male employees aged 45.3 +/- 6.8 years (mean +/- SD), all on unrestricted diet and on no pharmacological treatment were studied. Statistical associations between Na-Li countertransport activity and the hormonal factors being studied were investigated, with control for the possible confounding effects of age, BP and sodium intake. Na-Li countertransport was significantly and positively associated with plasma aldosterone concentration (r = 0.228, P0.01) but not with PRA or plasma ANP. Men in the highest quartile of the Na-Li countertransport distribution had significantly higher plasma aldosterone levels than men in the other quartiles (P0.01) and this difference was independent of age, body mass, PRA, urinary sodium excretion and blood pressure. It is concluded that plasma aldosterone levels are enhanced and disproportionately high relative to plasma renin activity in individuals with high Na-Li countertransport, independently of known potential confounders.

1. The present study was designed to investigate the relationships between circulating atrial natriuretic peptide, plasma and urinary cyclic GMP and sodium excretion under basal conditions and in response to changes in dietary sodium intake. 2. Measurements of plasma atrial natriuretic peptide and plasma and urinary (24 h collections) cyclic GMP, sodium and creatinine were made in (i) 30 normotensive subjects on their normal sodium intake and (ii) 12 subjects on the 5th day of a low and on the 5th day of a high sodium intake. 3. Plasma cyclic GMP, urinary cyclic GMP and fractional excretion of cyclic GMP in 30 normotensive subjects on their normal sodium intake were (means ± SEM) 5.4 ± 0.5 pmol/ml, 434.5 ± 31.8 pmol/min and 86.9 ± 8.6%, respectively. There were significant correlations between urinary cyclic GMP and its corresponding filtered load (r = 0.55) and between the renal clearance of cyclic GMP and that of creatinine (r = 0.44), but there were no significant associations between circulating atrial natriuretic peptide and plasma cyclic GMP or the fractional excretion of cyclic GMP or between urinary sodium and the fractional excretion of cyclic GMP. 5. Plasma atrial natriuretic peptide was significantly raised on the 5th day of the high sodium intake compared with the low sodium intake (10.6 ± 1.6 versus 4.2 ± 0.9 pg/ml; P 6. As neither plasma nor urinary cyclic GMP was strongly associated with circulating levels of atrial natriuretic peptide, the present study suggests that other factors may be more important than circulating atrial natriuretic peptide as determinants of extracellular cyclic GMP.

We have developed a radioimmunoassay for the measurement of immunoreactive BNP (1-32) in human plasma. Simultaneous measurement of ANP have also been carried out to allow for direct comparison between circulating BNP and ANP. Plasma levels of immunoreactive BNP (means +/- SEM) were 1.1 +/- 0.1 pmol/l in 36 normal healthy subjects and were significantly elevated in 50 patients with essential hypertension (1.6 +/- 0.2 pmol/l, P0.02). Similarly, in patients with essential hypertension plasma levels of ANP were also significantly raised (5.5 +/- 0.6 pmol/l, P0.001) when compared with the group of normal healthy subjects (2.8 +/- 0.2 pmol/l). ANP was significantly higher than BNP in normal subjects and in patients with essential hypertension, with ANP/BNP ratios of 2.8 +/- 0.2 and 3.8 +/- 0.3, respectively, in these two groups. A major finding was a significant and positive association between plasma levels of both BNP and ANP within the healthy subjects (r = 0.49, P0.05, n = 36) and within the hypertensive subjects (r = 0.76, P0.001, n = 50). When all plasma values for BNP and ANP were taken together for both groups, there was an overall correlation coefficient of 0.65 (P0.001, n = 86). Both BNP and ANP had significant positive associations with age in hypertensive patients, with correlation coefficients of 0.53 (P0.001, n = 50) and of 0.53 (P0.001, n = 50) for BNP and ANP, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of mouse peritoneal mast cells and mouse bone marrow-derived cloned mast cells with the protein synthesis inhibitor cycloheximide led to a marked abrogation of serotonin (5-hydroxytryptamine; 5-HT) release induced by a range of activators including antigen, anti-immunoglobulin E (anti-IgE) antibody, calcium ionophore A23187, and the polycation polylysine. Significant inhibition (28%) of IgE-mediated secretion was attained after incubation of peritoneal cells for only 2 hr, and inhibition progressed over a 24 hr period, reaching greater than 80% after 15 hr. When peritoneal mast cells were exposed to cycloheximide and then washed and returned to culture conditions, a substantial recovery of responsiveness to anti-IgE was seen after 5 hr. Under the same conditions to those used in functional studies, cycloheximide inhibited protein synthesis, measured as incorporation of [35S]-methionine, by purified peritoneal mast cells and cloned mast cells to 18.5% and 7.9% of control levels, respectively. These results show that synthesis of new protein over a period of a few hours is required to render mast cells fully responsive to stimuli that act via the IgE receptor, and to certain other stimuli that are receptor-independent.

OBJECTIVES (1) To compare urinary guanosine 3',5' cyclic monophosphate (cyclic GMP) excretion between normotensive subjects and essential hypertensive patients; (2) to determine the influence of changes in sodium intake on urinary cyclic GMP excretion in response to the neutral endopeptidase inhibitor candoxatril in essential hypertensives. DESIGN (1) Twenty-five normotensive subjects and 25 patients with established essential hypertension not on treatment; (2) Single oral dose of candoxatril in eight patients with essential hypertension after equilibration on a low- or high-sodium diet in a placebo-controlled, double-blind, randomized, crossover study. METHODS Blood pressure was measured by ultrasound sphygmomanometry. Atrial natriuretic peptide (ANP) and urinary cyclic GMP were measured by radioimmunoassay. Group comparisons were made using unpaired t-tests and two-way analysis of variance. RESULTS Plasma ANP was significantly raised in patients with essential hypertension compared with the normotensive group, but there was no difference in urinary cyclic GMP excretion. Plasma ANP increased significantly on the high- compared with low-sodium diet. After candoxatril, there were significant diet-related increases in plasma ANP and urinary sodium excretion up to 6 h after drug administration. There were similar increases in urinary cyclic GMP excretion on both diets, but there were no consistent differences in this excretion between the low- and high-sodium diets. CONCLUSIONS These observations not only point to the importance of ANP-cyclic GMP coupling as a determinant of the natriuretic response to endopeptidase inhibition, but also suggest that the excretion of urinary cyclic GMP can be influenced by other factors in addition to circulating ANP.

The identification of the atrial natriuretic peptides (ANP) as a new hormonal system has provided a new perspective on the mechanisms controlling renal sodium excretion and abnormalities in sodium homeostasis. The present article focuses on the potential importance of ANP (ANF 99–126) in essential hypertension with particular reference to circulating ANP levels and the relationship between the ANP and the renin–angiotensin system in the control of sodium balance and blood pressure. There is now considerable evidence demonstrating that a substantial proportion of patients with essential hypertension have raised circulating ANP levels. Given the known biological actions of ANP, these raised levels point to important compensatory mechanisms. This is further supported by studies during alterations in dietary sodium intake, as sodium restriction highlighted important relationships between ANP and the renin angiotensin system. The potential importance of ANP in essential hypertension is strengthened by recent demonstration of natriuretic and antihypertensive actions associated with small increases in circulating ANP as induced by administration of exogenous ANP. Furthermore, the recent development of orally active inhibitors of ANP metabolism now provides a basis to determine the therapeutic importance of specific manipulation of endogenous ANP levels in patients with essential hypertension.Key words: ANF, essential hypertension, sodium balance.

purpose: To assess the changes in sodium excretion and sodium balance after initiation of nifedipine treatment and after withdrawal of nifedipine. patients: Eight patients with uncomplicated mild to moderate essential hypertension were entered in a single-blind, placebo-controlled study of 39 days' duration. methods: Two 7-day periods while on a fixed sodium intake of 150 mmol/day approximately 3 weeks apart. After 4 days of a placebo and fixed sodium intake, patients were given nifedipine GITS (gastrointestinal therapeutic system) once a day and carefully studied for the following 4 days. Thereafter, patients continued to receive nifedipine GITS, and approximately 3 weeks later they were studied again for a week while on a fixed sodium intake. Nifedipine administration was stopped and changes occurring after withdrawal were studied. results: Nifedipine caused a significant increase in sodium excretion with a cumulative loss of sodium of 38 mmol per subject within the first 4 days of treatment. The withdrawal of nifedipine treatment caused a significant decrease in sodium excretion and a cumulative retention of sodium of 42 mmol per subject within the first 4 days of withdrawal. conclusions: Nifedipine causes an acute and a sustained reduction in sodium balance in patients with essential hypertension. This prolonged effect may contribute to the mechanism whereby nifedipine lowers blood pressure.

We studied the effect of amlodipine, a long-acting dihydropyridine calcium antagonist, on blood pressure, urinary sodium excretion, plasma renin activity, aldosterone and atrial natriuretic peptide in six patients (aged 47-63 yrs) with essential hypertension. Patients were placed on a fixed sodium intake of 150 mmol/day. After a control period, amlodipine 10 mg/day was given for two weeks. There was a gradual reduction in supine BP over the first two days of treatment, from 165/103 +/- 5/4 mmHg to 137/92 +/- 6/4 mmHg (P less than 0.001) and BP remained at this level during treatment. Three days after amlodipine was stopped the BP was still reduced at 136/87 +/- 5/4 mmHg but was back to pretreatment levels two weeks later. Plasma amlodipine rose after two weeks of treatment to 29.7 +/- 4.7 ng/ml but had only decreased to 15.0 +/- 3.4 ng/ml three days after the treatment was withdrawn. During the first two days of treatment there was no evidence of an increase in urinary sodium excretion and when amlodipine was withdrawn there was no evidence of sodium retention. Plasma renin activity increased from 1.26 +/- 0.30 to 2.99 +/- 0.68 ng/ml/h (P less than 0.001) and plasma atrial natriuretic peptide fell from 19.3 +/- 7.0 to 11.4 +/- 3.8 pg/ml (P less than 0.03) with two weeks of treatment. This study demonstrates that amlodipine is a long-acting calcium antagonist with a slow onset of action and a slow end of action after withdrawal. This makes it difficult to detect alterations in sodium balance when assessed by changes in urinary sodium excretion. However, one explanation for the increase in plasma renin activity and fall in atrial natriuretic peptide is a small reduction in total body sodium.

1. Aldosterone is suppressed by sodium loading. We studied the contribution of this decrease in plasma aldosterone to the natriuresis after acute sodium loading in healthy volunteers. 2. Two litres of saline [0.9% (w/v) NaCl] were infused during the second hour of a 6 h infusion of aldosterone (3 pmol min−1 kg−1) or placebo in eight healthy young men. On the placebo day, plasma aldosterone decreased by 30 min after the start of saline infusion and remained suppressed. During aldosterone infusion, plasma aldosterone was maintained at around 400 pmol/l. 3. Urinary sodium excretion, lithium clearance and plasma atrial natriuretic peptide increased and plasma renin activity decreased after saline infusion, whether or not aldosterone was infused. However, from 60 to 240 min after saline infusion, natriuresis was significantly less during aldosterone infusion than on the placebo day. In addition, saline loading led to a progressive increase in the ratio of sodium clearance to lithium clearance, used as an index of the fractional distal tubular rejection of sodium, and in the ratio of urinary sodium to potassium. These increases were prevented by the infusion of aldosterone. 4. This study suggests that there are differences in the mechanisms determining the early and the later responses to an acute sodium load. Suppression of aldosterone may explain much of the later increase in natriuresis after saline infusion. In addition, the results are consistent with a role for atrial natriuretic peptide in the immediate increase in sodium excretion after saline loading.

The associations between renal tubular sodium handling and plasma levels of atrial natriuretic peptide, renin activity and aldosterone were studied in 295 untreated men under normal living conditions. The renal clearance of ingested lithium was used as a marker of proximal tubular sodium handling. Plasma atrial natriuretic peptide was inversely related to creatinine clearance (r = -0.148, P less than 0.01) and directly and significantly related to the overall fractional excretion of sodium (r = 0.213, P less than 0.001) and to distal (r = 0.151, P less than 0.01) fractional sodium excretion. Plasma renin activity was inversely related to sodium excretion at both proximal (r = -0.145, P less than 0.05) and distal (r = -0.236, P less than 0.001) tubular site, whereas plasma aldosterone was significantly and inversely related to distal sodium excretion only (r = -0.305, P less than 0.001). The association between plasma atrial natriuretic peptide and distal sodium excretion in a large sample of men under normal living conditions supports the view of a possible tubular effect of the hormone of the overall control of sodium excretion in man.

N-Terminal pro ANP (atrial natriuretic peptide) in human plasma has been measured by radioimmunoassay after extraction on Sep-Pak cartridges. Immunoreactive N-terminal pro ANP circulates in human plasma at higher levels than alpha-hANP (approximately 20-fold higher in normal subjects) and was elevated in patients with essential hypertension, cardiac transplantation and patients with chronic renal failure. In chronic renal failure patients undergoing hemodialysis, C-terminal ANP (ANP 99-126), but not N-terminal ANP, declined significantly after dialysis. Gel filtration experiments demonstrated a single peak of N-terminal ANP immunoreactivity, eluting in parallel with synthetic human pro ANP 1-67, indicating a similar molecular size and the absence of low molecular weight N-terminal fragments.

The present study examines the responses of plasma atrial natriuretic peptide (ANP), aldosterone and plasma renin activity to small alterations in dietary sodium intake. Six normotensive subjects were equilibrated on a low sodium intake of 10 mmol/day for 4 days. Dietary sodium intake was then increased gradually by 50 mmol/day to a maximum of 350 mmol/day over a 7 day period. With the gradual increase in sodium intake there were progressive increases in urinary sodium and cumulative sodium balance. These were associated with gradual increases in plasma ANP and reductions in both plasma aldosterone and plasma renin activity. During the study there were no significant changes in blood pressure, urinary potassium and creatinine clearance. This study demonstrates a marked sensitivity of the responses of both the ANP and the renin-aldosterone system to small changes in sodium intake and points to their importance in the renal adaptations to small alterations in dietary sodium intake.

Plasma levels of immunoreactive N-terminal ProANP have been measured in plasma from 19 healthy individuals, 15 patients with essential hypertension, 8 cardiac transplant recipients and 8 patients with chronic renal failure using two separate radioimmunoassays (RIAs), one directed against ProANP (1-30) and the other against ProANP (79-98). The mean concentrations of ProANP (1-30) and ProANP (79-98) were elevated in these groups of patients. There were positive correlations between levels of ProANP (1-30) and ProANP (79-98), with a correlation coefficient of 0.97 (P less than 0.001, n = 50). In healthy individuals a 2-1 (isotonic) saline infusion significantly increased both ANP (99-126) (P less than 0.05, n = 8) and N-terminal ProANP (P less than 0.005, n = 8) within 15 min of the end of the infusion. Plasma N-terminal ProANP levels were still significantly elevated after 75 min (P less than 0.05, n = 8) and 225 min (P less than 0.05, n = 8), by contrast ANP (99-126) had returned to basal values. Gel filtration of plasma extracted on Sep-Pak C-18 from normal individuals and patients gave a single immunoreactive peak for N-terminal ProANP as measured by both N-terminal ProANP assays, indicating an absence of small N-terminal fragments and the presence of a single high molecular weight form. These studies demonstrate that the major circulating N-terminal ANP in man is probably ProANP (1-98) and that it is cosecreted with ANP (99-126).

1. To study the importance of cardiac innervation in the regulation of atrial natriuretic peptide, plasma atrial natriuretic peptide levels were measured during symptom-limited, graded exercise on a cycle ergometer in seven male orthotopic cardiac transplant recipients. 2. Resting plasma atrial natriuretic peptide was significantly higher in the transplant recipients than in two control groups, one matched to the transplant recipients (group 1) and the other to the age of the donor heart (group II). 3. The response to exercise of the cardiac transplant recipients was compared with the response of control group II. Mean maximal work load achieved with exercise was around 40% lower in the cardiac transplant recipients. During exercise, plasma atrial natriuretic peptide levels increased in both the cardiac transplant recipients and the control subjects. The increase in plasma atrial natriuretic peptide with exercise was greater in absolute, but less in percentage, terms in transplant recipients than in the control subjects. 4. The increase in mean arterial pressure with exercise was similar in patients and in control subjects; however, heart rate increased in the patients by only 33% compared with a rise of 151% in the control group. 5. These results provide insight into the control of the sodium regulatory hormone atrial natriuretic peptide. First, factors other than a change in heart rate appear of importance in the regulation of atrial natriuretic peptide. Secondly, these findings suggest that cardiac innervation is not of dominant importance in the modulation of atrial natriuretic peptide secretion.

Brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and N-terminal ANP are good research indices of the severity of heart failure. The stability of these peptides at room temperature has become an important factor in assessing their use as indicators of cardiac function in routine clinical practice. Inhibitors such as aprotinin are routinely added in the blood collection process, but may provide no benefit in sample collection and routine clinical practice. We assessed the stability of BNP, ANP and N-terminal ANP in blood samples collected in either the presence or the absence of the protease inhibitor aprotinin. Blood, either with or without aprotinin, was processed immediately (initial; 0 h) and after blood samples had been left for 3 h, 2 days or 3 days at room temperature. These times were chosen to reflect processing in a hospital outpatient clinic (2-3 h), or when posted from general practice (2-3 days). Initial plasma BNP, ANP and N-terminal ANP levels in the absence of aprotinin were 28.2+/-5.4, 44.2+/-7.9 and 1997+/-608 pg/ml respectively, and were not significantly different from initial values in the presence of aprotinin (29.0+/-5.9, 45.2+/-8.0 and 2009+/-579 pg/ml respectively). After 3 h at room temperature, there was a significant fall in ANP in the absence of aprotinin (36. 7+/-7.9 pg/ml; P0.005), but not in the presence of aprotinin (41. 2+/-7.6 pg/ml). Both BNP and N-terminal ANP were unchanged in either the absence (BNP, 27.6+/-5.5 pg/ml; N-terminal ANP, 2099+/-613 pg/ml) or the presence (BNP, 29.4+/-5.6 pg/ml; N-terminal ANP, 1988+/-600 pg/ml) of aprotinin. After 2 days at room temperature, ANP had fallen significantly in both the absence (16.9+/-3.4 pg/ml) and the presence (24.0+/-5.0 pg/ml) of aprotinin compared with initial values, and there was a significant difference in ANP levels in the absence and presence of aprotinin (P0.001). ANP levels had decreased further after 3 days at room temperature, to 11.9+/-3.4 pg/ml (no aprotinin) and 20.3+/-5.0 pg/ml (aprotinin added); these values were significantly different (P=0.002). In contrast, there was no change in the levels of BNP or N-terminal ANP after 2 or 3 days at room temperature, in either the absence or the presence of aprotinin. These studies indicate that aprotinin adds little benefit to the stability of cardiac peptides at room temperature. Blood samples for BNP and N-terminal ANP measurement used as a test of heart function in hospital clinics and by general practitioners in the community could be taken into blood tubes containing only EDTA as anticoagulant and without the additional step of adding the routinely used inhibitor aprotinin.

Perindopril is a new orally active non-sulphur containing inhibitor of angiotensin converting enzyme (ACE) with enzyme inhibition being due to perindoprilat, the active diacid [1, 2]. Previous work in patients with essential hypertension has demonstrated significant reductions in blood pressure with doses of 4-8 mg/day over a i month period [3, 4]. We have now investigated the effects of perindopril in essential hypertension not only on blood pressure but also on the activity of the renin-angiotensinaldosterone system as well as on plasma atrial natriuretic peptide (ANP). This study was approved by the local Ethical Committee, informed consent was obtained in every case and was carried out in 12 patients with uncomplicated essential hypertension. There was a 2 week runin period followed by a 2week placebo observation phase, all patients were then randomised to a 1 month cross over treatment period on 2 mg or 8 mg perindopril per day. At the end of the placebo and at the end of each treatment period measurements were taken at 2, 4, 6 and 24 h after taking the last tablet. Blood pressure and heart rate were measured with standardised ultrasound sphygmomanometer techniques. Blood was taken with the patient sitting upright for 10 min for plasma renin activity (PRA), aldosterone and ANP [5]. Plasma levels of perindopril and perindoprilat were measured by radioimmunoassay after separation on Dowex AGI x 2 ion exchange chromatography. Plasma ACE activity was measured as described [6] using hippuryl histidyl leucine as the substrate. Statistical analysis was by analysis of variance with repeated measures and with paired t-tests. Results are given as means with (SEM). Plasma levels of perindopril were dose -dependent, highest at 2 h after the oral dose and declined rapidly thereafter; by contrast, plasma level of perindoprilat increased rapidly and remained elevated up to 6 h after the oral dose. After perindopril there was marked dose and time-dependent (ANOVA P < 0.0001) inhibition of converting enzyme activity with maximal inhibition 4-6 h after perindopril well in agreement with the levels of perindoprilat (Table). As found with other ACE inhibitors, administration of perindopril was associated with time-dependent increases in PRA with highest levels at 4 h after perindopril for both doses (ANOVA P < 0.005), by contrast, there were reductions in plasma aldosterone (Fig. 1). There were no effects on plasma ANR During the placebo period, average blood pressures (supine) ranged from 159-163/98-106mmHg. After perindopril there were significant time-dependent changes for both doses of perindopril (ANOVA P = 0.015) with blood pressures being lower at 4 h than at 24 h after perindopril (4 h: 148 (4.4) 96 (2.6) for the 2 mg and 142 (5.3) 91 (3.4) mmHg for the 8 mg dose; 24 h: 155 (4.7) 102 (3.6) for the 2 mg and 154 (4.7) 97 (3.8) m m H g for the 8 mg dose respectively). Although there was a trend for lower blood pressures with the higher dose of 8 mg there was no overall statistically significant dose

Objective To examine the changes in plasma brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and blood pressure in patients with essential hypertension on a low, normal and high sodium intake. Design and methods Twelve patients with mild-to-moderate essential hypertension were studied. Plasma, urinary and blood pressure measurements were made with the patients on their usual sodium intake, then on the fifth day of a low (10 mmol/day) and on the fifth day of a high (350 mmol/day) sodium intake, the sequence being allocated randomly. Results Plasma levels of BNP and ANP increased significantly on the high sodium intake compared with when the patients were on their normal diet. The mean blood pressure on the high sodium intake was not significantly different from that with the patients on their normal diet. In contrast, plasma BNP and ANP decreased on the low sodium intake, but were not significantly different compared with when the patients were on their normal diet. However, there was a significant reduction in the mean blood pressure on the low sodium intake compared with when the patients were on their normal diet. Compared with the normal diet, BNP and ANP plasma levels showed similar percentage decreases on the low sodium intake and similar percentage increases on the high sodium intake. Conclusions These findings suggest that BNP and ANP are released in response to a common stimulus during changes in dietary sodium intake. The changes in plasma BNP and ANP observed with sodium restriction and sodium loading indicate the potential importance of BNP and ANP as a dual peptide system contributing to the maintenance of sodium balance and blood pressure regulation in patients with essential hypertension, during changes in dietary sodium intake.