Reciprocal Regulation of Plasma Apelin and Vasopressin by Osmotic Stimuli

Apelin is a neuropeptide that co-localizes with vasopressin (AVP) in magnocellular neurons and is involved in body fluid homeostasis. Osmotic stimuli have opposite effects on the regulation of apelin and AVP secretion in animal models, but whether this is true in humans is unknown. This study investigated the relationship among osmolality, apelin, and AVP in 10 healthy men after infusion of hypertonic saline or loading with water to increase and decrease plasma osmolality, respectively. Increasing plasma osmolality was accompanied by a parallel, linear increase in plasma AVP concentration and by a decrease in plasma apelin concentration. In contrast, decreasing plasma osmolality by water loading reduced plasma AVP concentration and rapidly increased plasma apelin concentration. These findings suggest that regulation of apelin secretion contributes to the maintenance of body fluid homeostasis. The osmotic pressure of body fluids is maintained within a remarkably narrow range in healthy adults. Body fluid homeostasis depends on neuronal pathways bearing very sensitive osmoreceptors,1 located along the lamina terminalis, including the circumventricular organs, such as the subfornical organ and the organum vasculosum of the lamina terminalis as well as the median preoptic nucleus.2 The subfornical organ and organum vasculosum of the lamina terminalis are neuronally interconnected with each other as well as with the median preoptic nucleus and the hypothalamic paraventricular and supraoptic nuclei.3 These neuronal pathways convert small changes in osmolality into a neuronal signal to neurons that influence sensations of thirst and systemic arginine vasopressin (AVP) release,2 thereby adjusting the intake or output of water to counteract changes in solute concentration.4,5 A recently discovered peptide, apelin, may also play a major role in the maintenance of body fluid homeostasis. Apelin, initially isolated from bovine stomach extracts,6 is the endogenous ligand of the human orphan G protein–coupled receptor APJ (putative receptor protein related to the angiotensin receptor AT1).6,7 It is a 36–amino acid peptide (apelin 36) derived from a single 77–amino acid precursor, proapelin.6,8,9 Proapelin has a fully conserved C-terminal 17–amino acid sequence, apelin 17 (K17F), including the pyroglutamyl form of apelin 13 (pE13F). K17F and pE13F both are present in rat brain and plasma,10 and apelin 36 is present in testis and uterus.11 All peptides exhibit a high affinity for the human8,12,13 and the rat apelin receptors.14 Apelin possesses various cardiovascular functions (for reviews,15–17). Apelin and its receptor have been detected in the endothelial cells of large conduit arteries, coronary vessels, and the endocardium of the right atrium.18–20 Apelin injection decreases BP in animals,9,21–23via nitric oxide production.21 Apelin has a positive inotropic effect both on isolated perfused rat hearts ex vivo24 and on normal and postmyocardial infarction rat hearts in vivo.25 Apelin administration in mice reduces left ventricular preload and afterload and increases contractile reserve and cardiac output.26 Finally, apelin-deficient mice develop heart failure with aging.27 Apelin and its receptor both are widely distributed in the brain9,14,28,29 but are particularly abundant in the supraoptic nucleus and paraventricular nucleus, where they co-localize with AVP in magnocellular neurons.10,29–31 Intracerebroventricular injection of K17F inhibits the typical phasic firing pattern of AVP neurons in lactating rats, resulting in decreased systemic AVP release and increased aqueous diuresis.10 Moreover, water deprivation in rats while increasing systemic AVP release and causing depletion of hypothalamic AVP stores decreases plasma apelin concentrations and results in hypothalamic accumulation of the peptide. Thus, the two peptides are conversely regulated to optimize AVP release into the blood circulation and prevent additional water loss through the kidney.10 Whether such opposite regulation of apelin and AVP secretion in response to osmotic stimuli is found in humans remains unknown. The aim of this clinical investigation was to examine the relationship between plasma osmolality, plasma apelin, and plasma AVP in healthy adults in various states of hydration. Hydration was modified by administration of a hypertonic saline infusion and by water loading to increase and decrease plasma osmolality, respectively. RESULTS Characterization of the Molecular Forms of Apelin Present in Human Plasma Apelin immunoreactivity (apelin-IR) was resolved as two major components eluting in fraction 28 and fractions 31 to 32 and a minor component eluting in fraction 40 (Figure 1). Calibration of the C4 column with apelin fragments revealed that the prominent immunoreactive compounds co-eluted with pE13F and K17F, respectively, and that the apelin-IR present in fraction 40 exhibited the same retention time as synthetic apelin 36 (Figure 1). Plasma Apelin Levels in Physiologic Unrestricted Water Intake Conditions on Day −1 At 8:00 a.m. on day −1, the plasma apelin concentration in 10 healthy individuals was 477 ± 167 fmol/ml; plasma osmolality (286 ± 4 mOsm/kg H2O) and plasma AVP concentration (median [interquartile range (IQR)] 1.8 [1.4 to 3.0] fmol/ml) both were within the physiologic range. Plasma osmolality, apelin, AVP, and apelin/AVP ratio remained stable between 8:00 a.m. and 8:00 p.m. with no significant changes associated with the 12-h overnight fluid and food restriction (Table 1). The median (IQR) within-subject variability in plasma apelin concentration as assessed by testing plasma samples collected between 8:00 a.m. on day −1 to 9:00 a.m. on day 1 was 18.6% (13.3 to 23.6%) and that of plasma AVP concentration was 23.3% (18.2 to 36.8%). There was no significant difference in plasma apelin or in plasma AVP concentrations between the individuals assigned to the hypertonic saline infusion on day 1 (group 1) and those assigned to the water loading test on day 1 (group 2) at any time point between 8:00 a.m. on day −1 and 9:00 a.m. on day 1 (Table 1). The plasma apelin/AVP ratio was also similar between the two groups (Table 1). There was no significant correlation between plasma apelin concentration and plasma AVP concentration in the absence of osmotic challenge (data not shown). Although plasma osmolality levels significantly differed between the two groups (Table 1), probably because group 1 and group 2 were studied sequentially at different times of the year (see the Concise Methods section), there was no significant correlation between plasma apelin or plasma AVP concentrations and plasma osmolality in the absence of osmotic challenge (data not shown). Hypertonic Saline Infusion in Group 1 The hypertonic saline infusion induced a smooth and linear increase in plasma osmolality from 288 ± 1.6 mOsm/kg H2O at baseline to 302 ± 3.1 mOsm/kg H2O at the end of the infusion (P < 0.0001 versus baseline; Figure 2A) and in plasma sodium (Na+) concentration (P < 0.0001 versus baseline; Table 2), associated with a parallel linear increase in plasma AVP concentration from 2.6 (2.4 to 3.5) fmol/ml at baseline to 10.0 (8.9 to 11.3) fmol/ml at the end of the infusion (P < 0.0001 versus baseline; Figure 2B). There was a significant and marked decrease in hematocrit (P = 0.0017), total protein concentration (P = 0.0003), and plasma active renin (P = 0.0019) and aldosterone concentrations (P = 0.0198) associated with an increase in the estimated extracellular fluid volumes (ECFV; 17.4 ± 1.6%; Table 2). There was a highly significant positive linear correlation between plasma AVP concentration and plasma osmolality between baseline and time 120 min (r = 0.91, n = 35, P < 0.0001; Figure 3A), and the extrapolated median osmotic threshold for AVP secretion was 283.1 mOsm/kg H2O (range 278.4 to 285 mOsm/kg H2O). The time course of plasma apelin concentration was biphasic. During the first 80 min of the hypertonic saline infusion, the plasma apelin concentration decreased linearly from 567 ± 124 fmol/ml at baseline to 326 ± 111 fmol/ml (P < 0.01 versus baseline; median change from baseline [IQR] −43.8% [−47.2 to −20.4]; Figure 2C), contrary to the plasma AVP concentration. Simple linear regression of plasma apelin and plasma osmolality was thus applied to each individual infusion of hypertonic saline between 0 and 80 min, the linear part of the decrease in plasma apelin with time. There was a highly significant negative linear correlation between plasma apelin concentration and plasma osmolality for the period 0 to 80 min (r = −0.70, n = 25, P = 0.0004; Figure 3B). There was also a significant negative linear correlation between plasma apelin concentration and plasma AVP concentration for the period 0 to 80 min (r = −0.52, n = 25, P = 0.015; Figure 3C); however, after adjustment for plasma osmolality, the relationship between plasma apelin concentration and plasma AVP concentration was no longer significant (r = −0.31, n = 25, P = 0.3806; data not shown). This indicates that the correlation between plasma apelin and AVP concentration observed during the first 80 min of the hypertonic saline infusion was driven by the plasma osmolality. During the last 40 min of the hypertonic saline infusion, plasma apelin concentration increased from 326 ± 111 fmol/ml at 80 min to 458 ± 136 fmol/ml at 120 min (P = 0.4961 versus baseline; Figure 2C). The plasma apelin/AVP ratio decreased massively from 196 (193 to 202) fmol/fmol at baseline to 47 (30 to 50) fmol/fmol at time 80 min (P < 0.0001 versus baseline) and remained at a low value up to 120 min (52 [46 to 52] fmol/fmol; P < 0.0001 versus baseline; Figure 4). To search for an explanation for the rise in plasma apelin after its initial decrease, we used the individual equations of the regression line between plasma apelin and plasma osmolality between baseline and time 80 min to predict a curve for plasma apelin between time 0 and time 120 min if plasma osmolality was the sole determinant factor influencing plasma apelin levels. The predicted curve was plotted and compared with the observed curve (Figure 5A). The two curves diverged from time point 80 min to time point 120 min (end of the infusion). We then plotted the absolute difference between the observed and the predicted apelin values at each time point (Figure 5B). From 80 min to the end of infusion, the difference between the observed and the predicted apelin values increased linearly. We then superimposed the time course of the relative changes in estimated ECFV from time 0 to time 120 min (Figure 5B). The observed plasma apelin concentration started to diverge from the predicted value (i.e., the difference diverged from 0) when the estimated ECFV had increased by approximately 12 to 15%. The expansion in the estimated ECFV had no apparent effect on plasma AVP concentrations (see Figure 2B). Water Loading in Group 2 Plasma osmolality decreased rapidly and significantly from baseline (284.6 ± 1.8 mOsm/kg H2O) to a nadir at 60 min (change from baseline −4.0 mOsm/kg H2O; 95% confidence interval −7.0 to −1.0 mOsm/kg H2O; P = 0.0076) then progressively increased to reach the baseline value at 240 min (Figure 6A). The plasma Na+ concentration decreased simultaneously (P = 0.0033 versus baseline; Table 2). There was a parallel significant decrease in plasma AVP concentration from 1.9 (1.8 to 2.1) fmol/ml at baseline to a nadir of 1.5 (1.4 to 1.7) fmol/ml (P = 0.0139 versus baseline; median variation from baseline of −30.4% [IQR −45 to −28.6]) followed by a slight increase by 240 min (Figure 6B). Water loading had no significant effect on hematocrit or total protein, plasma active renin, and aldosterone concentrations, indicating no significant change in estimated ECFV (Table 2). In contrast to plasma AVP, the plasma apelin concentration increased from 335 ± 83 to 527 ± 187 fmol/ml (P = 0.0047 versus baseline; median variation from baseline IQR 73.9% 23.4 to 74.5) immediately after water loading (time 30 min; Figure 6C); it then remained stable at approximately 530 fmol/ml for 210 min (time 240 min). The plasma apelin/AVP ratio increased massively from 144 (121 to 189) fmol/fmol at baseline to 424 (222 to 475) fmol/fmol immediately after water loading (P = 0.0007 versus baseline; Figure 4); it then remained stable at approximately 300 fmol/fmol for 210 min (time 240 min). DISCUSSION The main finding of this study is that acute osmotic stimuli induce opposite regulation of plasma apelin and AVP concentrations in humans, as previously reported in rodents.10,31 Apelin-IR was previously detected in human plasma using commercially available apelin-36 RIA or apelin-12 EIA kits,32–36 but the data revealed an important disparity in the concentrations of apelin circulating in plasma of healthy individuals (from 10 to 1000 fmol/ml).32–36 In addition, circulating apelin in human plasma has only been analyzed by gel filtration,37 a method that does not allow accurate characterization of the molecular forms of immunoreactive apelin. Here, we first identified the molecular forms of apelin present in human plasma in vivo by combining HPLC analysis with RIA detection. We showed for the first time that K17F and pE13F are the predominant forms of apelin present in human plasma and that the concentration of apelin 36 is much lower, consistent with previous findings in rodents.10 Using this assay, the physiologic concentrations of plasma apelin in 10 healthy lean male individuals on a normal sodium diet were 477 ± 167 fmol/ml on day −1 at baseline (8:00 a.m.) after an overnight fast. On a molar basis, plasma apelin concentration was physiologically 200 to 250 times higher than plasma AVP concentration (plasma apelin/AVP ratio: 233 [188 to 265] fmol/fmol) in the absence of any osmotic challenge. This may reflect the multiple sources of circulating apelin. Circulating apelin originates at least in part from magnocellular neurons10 but may also be produced by the gastrointestinal tract,38 adipocytes,34 and vascular and endocardial endothelial cells.19 We also observed that in conditions in which water intake was unrestricted and plasma osmolality remained stable within a narrow range, the plasma apelin concentration remained stable with a median within-subject variability of 18.6% (13.3 to 23.6%). It was of the same magnitude as that for plasma AVP concentration. The low within-subject variability contrasting with the high between-subject variability in plasma apelin concentrations may suggest some genetic influence in the regulation of apelin secretion, as it has been suggested also for AVP secretion.5,39 As expected,4,5 the increase in plasma osmolality induced by the hypertonic saline infusion was a potent physiologic stimulus for AVP release. The median threshold of AVP release was within the expected physiologic range, consistent with previous reports.4,5 Contrary to the changes in plasma AVP, the linear increase in plasma osmolality had the opposite effect on plasma apelin, the concentration of which declined linearly by 43.8% (20.4 to 47.2%) 80 min after the start of the during this the plasma apelin concentration was highly significantly and to plasma osmolality and to plasma AVP concentration. The initial decrease in plasma apelin concentration during the first 80 min of the hypertonic saline infusion was followed by a slight increase in plasma apelin concentration during the last 40 data suggest that the rise in the plasma apelin concentration after its initial decrease may be by the increase in ECFV its baseline) as also by the significant and marked decreases in total protein, and plasma active renin, and aldosterone In this ECFV expansion had no effect on plasma AVP changes in pressure are potent stimuli for AVP water decreased plasma osmolality and plasma Na+ and AVP The decrease in plasma osmolality was accompanied by a increase 30 in plasma apelin concentration to a the stimulus was and not mOsm/kg H2O; 95% confidence interval −7.0 to −1.0 mOsm/kg it was to the plasma apelin concentration at a for 210 min to contrasting with the opposite and decrease in plasma AVP concentration to The opposite changes in plasma apelin concentration after the increase or water decrease in plasma osmolality both were much than the within-subject variability in plasma apelin concentration. This is consistent with plasma osmolality a major physiologic of plasma apelin levels in humans as previously reported in rodents.10,31 We previously showed that injection of apelin AVP release in the in and lactating infusion of AVP induced apelin accumulation in magnocellular this effect is by a receptor Moreover, apelin response to in rats is opposite to that of water deprivation in rats reduces plasma apelin concentrations and an of the peptide in magnocellular AVP it increases plasma AVP concentrations and decreases AVP neuronal stores because AVP is than it is The increased release of AVP during in phasic of systemic AVP accumulation of apelin in the same neurons is thus not and the of apelin on AVP thus systemic AVP release. The opposite regulation of apelin and AVP during water deprivation in rats AVP and apelin to in to the systemic AVP release for a thus in water It is that results that the same osmotic stimulus to an opposite regulation of plasma AVP and apelin levels in This that apelin, AVP, may to the maintenance of body fluid homeostasis in humans as in Finally, the time course of plasma apelin/AVP ratio with changes in osmolality additional because baseline plasma AVP concentrations were approximately to 200 times than plasma apelin concentrations on a molar basis, the apelin/AVP ratio revealed significant during both the hypertonic saline infusion decrease from 196 to fmol/fmol) and the test increase from 144 to 424 Thus, the apelin/AVP ratio may the in the with low values indicating a and high values a Whether the plasma apelin/AVP ratio may be a than each in the of states remains to be in study the first in humans of the regulation of plasma apelin concentration by osmotic We also that this regulation is opposite to that of AVP, consistent with results in animal after water This that apelin, AVP, may in the maintenance of body fluid homeostasis in humans as in It is not whether the changes in the plasma apelin concentration by changes in osmotic pressure only reflect changes in secretion or have or effects on water to Apelin receptor has been detected in a of and in cells along the in the of the a of the a role in water and sodium It be of to plasma apelin levels in parallel with plasma AVP in various states of and the relationship between apelin AVP and osmolality in plasma may of the multiple of states of or concentration in Finally, the of of the apelin receptor be an or to receptor for the of water retention healthy male to ± 1.9 the study after and The was by the and the investigation was to of were to at the at a.m. on day −1 and remained for 36 intake day −1 was unrestricted 9:00 and were at and 8:00 p.m. approximately From 9:00 p.m. 9:00 a.m. on day from water and food At 9:00 a.m. on day after a 12-h overnight and water an infusion of a saline at the of min to increase plasma osmolality (group 1). The body water within 30 min to decrease plasma osmolality (group 2). In the two blood was at various times on −1 and 1 for plasma osmolality, apelin, AVP, active renin, and total protein with the for 1 each blood was collected and during the for of The two (group 1 and group 2) were and sequentially at two different of the year to for group 1 and to for group 2). group was its Methods Plasma osmolality was by The used for blood samples and for plasma AVP, active renin and aldosterone were as plasma apelin blood was into and at at for min and at HPLC human plasma samples were on a was with and the was reduced The was analyzed by HPLC on a C4 column with a of at a of 1 The concentration of in the eluting was to fractions were collected and for K17F, and apelin 36 used as were to the same conditions as human plasma apelin plasma samples were with of and of and at at for 10 The were collected and to with 12 and 2 The samples were then with 1 of acid with and a with of The were with of and apelin peptides were with 1.5 of The ± was ± The were and in of RIA apelin concentration was by RIA using highly the apelin K17F produced in the This two in K17F, of to pE13F and We thus used pE13F as a to to K17F and pE13F endogenous levels. using pE13F as a and with pE13F as with K17F, with and with apelin decreased with loss of from the part of K17F and was with fragments of K17F and various other including angiotensin angiotensin neuropeptide and Plasma samples were with and K17F to a total of and at the was and as The was and the was 12 The and of variation were and respectively. of human plasma samples of to the and curves that of pE13F used as a (data not shown). are as with IQR for to be distributed and for relative from other data are as ± were analyzed using for time with a within the time effect was were using the adjustment for multiple between were estimated using analysis of after of the P < was to be was used for HPLC analysis of apelin-IR in human A human plasma was on a C4 and the immunoreactive in the HPLC fractions was by This is of the of each apelin 30 ± K17F, ± and apelin ± The line indicates the The the of the of a hypertonic saline infusion on plasma osmolality and plasma AVP and apelin Plasma osmolality and apelin are as ± Plasma AVP is as The for time on day 1 with a within was significant for were using the adjustment for multiple time with baseline at 9:00 a.m. on day < < < versus baseline a.m. on day between plasma AVP concentration and plasma osmolality between plasma apelin concentration and plasma osmolality and between plasma apelin concentration and plasma AVP concentration during a hypertonic saline infusion of a hypertonic saline infusion min and a water within 30 min on the plasma apelin/AVP are as and The for time on day 1 was significant in each were using the adjustment for multiple time to baseline at 9:00 a.m. on day < < < versus baseline a.m. on day course of observed and predicted plasma apelin concentration during the hypertonic saline The of the regression line between plasma apelin and plasma osmolality between times 0 and 80 min was then used to predict the curve for plasma apelin between time 80 and time 120 of the absolute difference between the observed and the predicted plasma apelin values and of the relative increase in estimated extracellular fluid The observed and predicted values diverged estimated ECFV had increased by 12 to 15%. are ± of a water within 30 min on plasma osmolality and plasma AVP and apelin Plasma osmolality and apelin are as ± Plasma AVP is as median and The for time on day 1 was significant for were using the adjustment for multiple time with baseline at 9:00 a.m. on day < < < versus baseline a.m. on day Plasma apelin, osmolality, and AVP levels in physiologic unrestricted water intake conditions from day −1 (8:00 a.m.) to day 1 a.m.) in 10 healthy male and total protein changes during hypertonic saline infusion and water was by a from the and and the We the of the the and the healthy The of the renin, the and the AVP is much