Atrial natriuretic peptide-cyclic GMP relationships in normal humans: effects of dietary sodium intake

Transcription

1 Clinical Science (1993) 85, (Printed in Great Britain) 13 Atrial natriuretic peptide-cyclic GMP relationships in normal humans: effects of dietary sodium intake G. A. SAGNELLA, N. D. MARKANDU, M. G. BUCKLEY, D. R. ].'SINGER and G. A. MAcGREGOR Blood Pressure Unit, Department of Medicine, St George's Hospital Medical School, London, U.K. (Received 21 July 1992/7 October 1992; accepted 7 April 1993) 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 (24h 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 f SEM) 5.4 f 0.5 pmol/ml, f 31.8 pmol/ min and 86.9 f 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=o.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 f 1.6 versus 4.2 f 0.9 pg/ml; P< 0.05). Similarly, there were increases in urinary cyclic GMP excretion (692.3 & 43.4 versus f 41.9 pmol/min, P< 0.05), but there were no significant differences in the fractional excretion of cyclic GMP. 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. INTRODUCTION Cyclic GMP is now generally recognized as a second messenger for the biological actions of a- atrial natriuretic peptide (ANP). Extracellular cyclic GMP originates from intracellular cyclic GMP [l]. This raises the possibility of using the measurement of extracellular (plasma and urinary) cyclic GMP as a marker of the biological actions of ANP. Indeed, several studies in man have clearly demonstrated an association between increases in plasma ANP, plasma/urinary cyclic GMP and urinary sodium excretion during infusion of exogenous ANP [2-51 as well as during acute volume expansion [6-81, water immersion [9, lo] and immediately after recumbancy [ 113. Similarly, short-term increases in urinary cyclic GMP have also beeen demonstrated after administration of a neutral endopeptidase inhibitor [12, 131. Raised circulating levels of ANP and cyclic GMP have also been reported in patients with cardiac and renal disease [ However, as yet, little is known of the relationship between circulating ANP, plasma cyclic GMP and urinary cyclic GMP on a more longer term basis, particularly during increases in endogenous ANP such as in the adaptive responses to changes in dietary sodium intake [17, 181. The objectives of the present study, therefore, were to examine further the relationship between circulating ANP, plasma cyclic GMP and urinary cyclic GMP with particular reference to the origin of urinary cyclic GMP and the importance of ANPcyclic GMP coupling in the control of sodium excretion. This work has been published in part in abstract form [18aJ. METHODS Subjects Cross-sectional studies. Measurements were carried out in 30 normotensive healthy subjects (29 white, one Asian; 18 male; 12 female) on their usual sodium intake. The average age was 44 years (range years); the average weight of the subjects was 76.7 kg (range kg). Key words: atrid natriuretic peptide, cyclic GMP. sodium, urinary excretion. Abbreviations: ANP, a-atrid natriuretic peptide; GFR, glomerular filtration rate. Correspondencc: Dr G. A. Sqnella. Blood Pressure Unit, Department of Medicine, St George's Hospital Medical School, London SW17 ORE U.K.

2 ~ 14 G.A. Sagnella et al. Effects of dietary sodium alteration. Measurements were obtained in 12 normotensive subjects (seven male, five female; all white; age range years). Changes in sodium intake were carried out by dietary alterations [19]. Briefly, a low sodium intake was achieved with a diet containing 10mmol of sodium/day; for the high sodium intake, the low sodium intake was supplemented with 34 Slow sodium tablets/day (CIBA Laboratories, Horsham, Sussex, U.K.; 10mmol of sodium/tablet). The diets were given in a random order and all measurements were taken on the 5th day of the low and on the 5th day of the high sodium intake. Blood pressure Blood pressure (supine) was measured using an ultrasound sphygmomanometer (Arteriosonde; Roche) with an automatic recorder. Each measurement was taken as the mean value of five readings taken at 1-2 min intervals. Blood and urinary collections All blood samples were taken by venepuncture after the subjects has been sitting upright for 10min. All samples were taken between and hours. Plasma ANP was measured after extraction on Sep-Pak cartridges as described previously [20]. Blood for measurement of plasma cyclic GMP was collected in potassium-edta tubes, the blood was centrifuged and the plasma was stored at -20 C in a separate potassium-edta tube. Urinary measurements were made on 24h urine samples. Plasma and urinary electrolytes and creatinine were measured using standard methods. Table 1. Plasma and urinary cyclic GMP in 30 normotensive subjects on their normal sodium intake. Values are meanrksem. Plasma concn. (pmol/ml) Urinary excretion (pmol/min) Filtered load (pmol/min) Fractional excretion (%) Clearance (mllmin) Cyclic GMP 5.4 k k k f k 7.7 assay coefficients of variation were < 15% (n= 10). Average plasma cyclic GMP levels obtained using this method were well in agreement with our previously reported values using a method involving extraction and chromatography of plasma cyclic GMP [8]. Calculations and statistics Creatinine clearance (ml/min) was taken as a measure of glomerular filtration rate (GFR). The filtered load was calculated as the product of the plasma concentration multiplied by the GFR. Fractional excretion was calculated as the ratio of the urinary excretion rate divided by the filtered load and was expressed as a percentage. Group values are given as means+sem. Paired comparisons were carried out using paired t-tests. Regression lines were obtained by the method of least squares; correlation coefficients were calculated to test for associations between pairs of measurements. All statistical tests were carried out using the Statistical Package for the Social Sciences [21]. Measurement of urinary and plasma cyclic GMP Urinary cyclic GMP was measured by radioimmunoassay as described previously [13]. Plasma cyclic GMP was measured by radioimmunoassay after acetylation using commercially available reagents (Iodinated cyclic GMP assay kit; Amersham International plc, Slough, Berks., U.K.). On the day of the assay, plasma was thawed and then kept on ice. A 50p1 aliquot was added to 1.95ml of the acetate buffer provided with the assay kit which had been supplemented with EDTA (5 mmol/l). Acetylation (using 500p1 volumes of the diluted plasma/ standards) were then carried out according to the manufacturer s instructions. Dilution curves using plasma samples were consistent with those using cyclic GMP standard. The average recovery of synthetic cyclic GMP added to the plasma (four doses throughout the range of plasma levels) was 87.2%. The dose-response curve was effectively linearized using a log-logit transform and this was used to calculate cyclic GMP concentration in the unknown plasma samples. Within- and between- RESULTS Supine systolic and diastolic blood pressures in the 30 normotensive subjects were and 71 k 1 mmhg, respectively. Plasma ANP was pg/ml. Plasma and urinary cyclic GMP, as well as calculated filtered load, fractional excretion and clearance of cyclic GMP, are given in Table 1. By contrast with the amount of sodium excreted (fractional excretion 0.6 & average urinary cyclic GMP excretion was nearly 80% of the filtered load. Furthermore, the clearance of cyclic GMP (91.9 f 7.7 ml/min) was close to that of creatinine ( & 5.8 ml/min) and there was a significant correlation between the clearance of cyclic GMP and that of creatinine (r=0.44; P =0.01). There was also a significant association between urinary cyclic GMP excretion and the filtered load of cyclic GMP (r =0.55; P = 0.002; Fig. 1). No significant associations between plasma or urinary cyclic GMP (including fractional excretion) and the subjects age and sex were observed.

3 Cyclic GMP and endogenous atrial natriuretic peptide 15 I I I I I I lo Filtered load of cyclic GMP (pmol/min) Fig. 1. Urinary cyclic CMP excretion plotted against filtered load of cyclic GMP in 30 normotensive subjects Table 2. Plasma and urinary measurements on the 5th day of a low sodium intake and on the 5th day of a high sodium intake, Values are meansksem (n= 12). Statistical significance: *PcO.OS compared with the low sodium diet. Plasma ANP (pt/ml) Cyclic GMP (pmol/ml) Low sodium intake I High sodium intake 4.2k _+ 1.6; 3.6k f0.5 Urine Cyclic GMP (pmollmin) k f 43.4' Sodium (mmolj24h) 39.3 * f 22.3: Creatinine clearance (ml/min) I I I.2 k9.4 I 17.5 k6.3 Fractional excretion Cyclic GMP (%) Sodium (%) 140.9k k I O. I* Relationships between circulating ANP, cyclic GMP and urinary sodium excretion In this group of normotensive subjects, there was no significant correlation between plasma ANP and plasma cyclic GMP (r=0.14) or the fractional excretion of cyclic GMP (r=o.ll); the correlation coefficient between plasma ANP and urinary cyclic GMP was higher (r =0.33), but it did not reach statistical significance. Also, no significant associations were observed between urinary sodium (or the fractional excretion of sodium) and plasma cyclic GMP or the fractional excretion of cyclic GMP (results not shown). Effects of changes in dietary sodium intake Group values obtained on the 5th day of the low and on the 5th day of the high sodium intake are summarized in Table 2. Plasma ANP was approximately three times higher on the high sodium intake compared with the low sodium intake; there were relatively smaller (average 1.7-fold) but significant increases in 24h urinary cyclic GMP. Similarly, there were small increases in plasma cyclic GMP (1.5-fold), but these did not achieve statistical significance (P = 0.075). However, on the higher sodium intake, compared with the low sodium intake, there was a small, but significant, increase in the calculated filtered load of cyclic GMP (536.0k63.3 versus f44.4 pmol/min respectively; P = 0.03). There were no significant differences in the fractional excretion of cyclic GMP (Table 2). Average fractional excretion of cyclic GMP in this group of subjects was higher than that observed in the larger cross-sectional group who were studied on their normal sodium intake. This higher average value reflects a much higher individual variability in this group, in particular, several of the 12 subjects had fractional excretions greater than 100% on the low as well as on the high sodium intake. There was no significant association between the relative changes in plasma ANP from the low to the high sodium intake and the corresponding changes in urinary cyclic GMP or the fractional excretion of cyclic GMP (results not shown). DISCUSSION The present study was designed to investigate several interrelated aspects of the relationships between circulating ANP, cyclic GMP and urinary sodium excretion, and in particular: (i) the origin of renal cyclic GMP; (ii) the contribution of ANP to plasma and urinary cyclic GMP and (iii) the relationship between ANP-cyclic GMP and urinary sodium excretion during changes in dietary sodium intake. Both plasma cyclic GMP and urinary cyclic GMP (from 24 h collections) were measured in normotensive subjects. In one group, measurements were carried out on a cross-sectional basis with the subjects on their normal sodium intake; in the other group, measurements were carried out in response to changes in dietary sodium intake. Earlier studies [22] in humans using cyclic GMP infusion as well as radioactively labelled cyclic GMP demonstrated that plasma cyclic GMP was not protein-bound and was freely filtered at the glomerulus. The present observations on the renal handling of cyclic GMP also suggest that urinary cyclic GMP seems to originate from the plasma ultrafiltrate as: (i) there was a significant association between the clearance of creatinine and that of cyclic GMP and (ii) urinary cyclic GMP excretion was significantly related to the filtered load of cyclic GMP; however, there was considerable individual variability in the fractional excretion of cyclic GMP and, in some subjects, the fractional excretion was greater than 100%. This suggests that, to some extent, the kidneys may also contribute to the urinary excretion of cyclic GMP. Given that infusions of exogenous ANP are asso-

4 16 G.A. Sagnella et al. ciated with increases in plasma and urinary cyclic GMP [l-51, and that ANP is a potent stimulator of particulate guanylate cyclase [I], the association between ANP and extracellular cyclic GMP was subsequently investigated. However, in the crosssectional study there was no direct association between circulating ANP and plasma cyclic GMP, urinary cyclic GMP or the fractional excretion of cyclic GMP. The range of circulating ANP in the normotensive group investigated was relatively narrow and therefore this may provide one explanation for the lack of significant associations between circulating ANP and cyclic GMP. Bell et al. [Ill also observed an apparent dissociation between circulating plasma ANP and cyclic GMP during day-time sampling in ambulatory or seated normotensive subjects; interestingly, an association was observed when the subjects were supine, more than likely due to the immediate increase in ANP when adopting the supine position. In a previous study, Fujio et al. [IS] observed a relationship between plasma ANP and circulating cyclic GMP in normotensive subjects. However, the range of ANP was considerably higher than in the present study; furthermore, no information was provided on the renal handling of cyclic GMP. Since previous work has clearly demonstrated raised cyclic GMP during acute increases in circulating ANP such as during saline infusion [6-81 and after endopeptidase inhibition [12, 131, the present study was extended to examine the relationships between circulating ANP and plasma and urinary cyclic GMP in response to more chronic changes in endogenous ANP, such as those induced by changes in dietary sodium intake. Increases in dietary sodium intake in human subjects are associated with increases in circulating ANP [17, 181. In the present study measurements were taken after 5 days on a low and after 5 days on a high sodium intake. In agreement with previous studies there was an approximate three-fold increase in plasma ANP. However, although there was a small increase (average 1.7-fold) in urinary cyclic GMP, no changes in the fractional excretion of cyclic GMP were observed. The raised urinary cyclic GMP may well have been a consequence of a raised filtered load as the filtered load of cyclic GMP was significantly greater on the high sodium intake than on the low sodium intake. These results therefore would tend to support an association between these variables. But, in conjunction with the results of the cross-sectional studies, they suggest that, in general, the contribution of circulating ANP to plasma cyclic GMP and urinary cyclic GMP may be relatively small. Similarly, no significant associations were observed between urinary sodium excretion and urinary cyclic GMP excretion. In particular, although the fractional excretion of sodium increased nine-fold from the low to the high sodium intake, there were no significant changes in the fractional excretion of cyclic GMP (Table 2). These results do not necessarily exclude the possibility that ANP-cyclic GMP coupling is an important determinant of the changes in sodium excretion on a day-to-day basis. Certainly, from animal experiments, in rats in particular, there is considerable circumstantial as well as more direct evidence supporting the importance of cyclic GMP as a second messenger for the renal natriuretic actions of ANP (see [23] for a review). However, a dissociation between cyclic GMP excretion and urinary sodium excretion in response to ANP infusion has been observed in dog experiments [24]. To what extent these discrepancies reflect species differences in renal cyclic GMP handling remains unclear. Nevertheless, it is emphasized that although the observations documented, in the present study may merely reflect an insufficient systemic/renal overflow of intracellular cyclic GMP, another and possibly more likely explanation is the likelihood that other factors, apart from ANP, might also influence both plasma cyclic GMP and urinary cyclic GMP. Cyclic GMP is produced from activation of the particulate (ANP-sensitive) as well as the soluble guanylate cyclase [25]. Furthermore, intracellular cyclic GMP and thereby the amount released into the extracellular space could also be influenced by the cyclic GMP clearance/breakdown mechanisms, including cyclic GMP phosphodiesterases [25, 261. Phosphodiesterase activity is of particular interest as a determinant of plasma and urinary cyclic GMP excretion as recent studies [27, 281 have demonstrated marked increases in both circulating and urinary cyclic GMP after administration of a relatively specific inhibitor of cyclic GMP phosphodiesterase. Soluble guanylate cyclase is a distinct enzyme from the particulate ANP-sensitive enzyme and can be activated by several factors. Of these, endothelium-dependent relaxing factor-related factors are the most important [25, 261. Indeed, recent studies have demonstrated increases in urinary cyclic GMP in response to agents known to activate production of nitric oxide [29, 301. In summary, in normal human subjects, urinary cyclic GMP seems to originate from the plasma ultrafiltrate; however, a substantial proportion may also originate intrarenally, but neither plasma cyclic GMP, urinary cyclic GMP or the fractional excretion of cyclic GMP can be taken as a direct index of circulating ANP. These results do not, of course, exclude the importance of ANP in the control of sodium excretion, but merely highlight the possibility that, in normal human subjects, other factors may be more important than ANP as determinants of plasma and urinary cyclic GMP. ACKNOWLEDGMENTS We are grateful to Maxine Crane (SRN) and Christine Carney (SRN) for their assistance in these studies.

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