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Clinical Neuroendocrinology and Department of Internal Medicine, University of Lübeck, 23538 Lübeck, Germany
Address all correspondence and requests for reprints to: J. Born, Ph.D., Medizinische Universität zu Lübeck, Klinische Forschergruppe, Haus 23a, Ratzeburger Allee 160, 23538 Lübeck, Germany.
Abstract
Atrial natriuretic peptide (ANP) has been considered a potential candidate participating in the inhibitory control of pituitary-adrenal secretory activity. Here, we investigated the influence of ANP, infused at two different doses and over infusion intervals of two different durations, on the release of ACTH and cortisol after stimulation with CRH and with combined administration of CRH and vasopressin (VP). In young healthy men, three experiments were conducted. In Exp I, ACTH/cortisol secretory responses to CRH (50 µg) were examined during and after a 45-min period of ANP infusion at a rate of 4.4 µg/min (starting 15 min before CRH injection). In Exp II, ACTH/cortisol secretory responses to CRH (50 µg) were examined during and after a 90-min infusion period of ANP administered at rates of 4.4 and 8.8 µg/min. In Exp III, ANP was infused at a rate of 4.4 µg/min over 90 min, but instead of CRH, a combined administration of CRH (50 µg) and VP (0.5 IU infused within 5 min) was employed to stimulate ACTH/cortisol release. ANP diminished pituitary-adrenal secretory responses within the first hour after stimulation with exogenous secretagogues. Thereafter, the effect of ANP turned in the opposite direction, with distinctly enhanced concentrations of ACTH and cortisol during the third hour after stimulation. The inhibitory effect of ANP during the first hour of the pituitary-adrenal response was more pronounced on concentrations of cortisol than ACTH and was also more pronounced after combined administration of CRH/VP than after stimulation with CRH alone. Increasing the dose of ANP enhanced the late stimulatory effect on ACTH/cortisol release, thereby terminating the early period of inhibited ACTH/cortisol release more abruptly. The late stimulatory effect was enhanced with prolonged infusion of ANP. In addition, it was associated with reduced hematocrit, increased urine volumes collected, increased heart rate, and enhanced plasma VP concentrations. Together, these changes suggest that the late stimulatory effect of ANP on ACTH/cortisol release reflects an effect secondary to its hypovolemic actions. This stimulatory effect originating from peripheral systemic actions of ANP after exogenous administration appears to override a more direct inhibitory action of the peptide on pituitary-adrenal secretory activity. Therefore, we would expect that with localized release into portal hypophyseal blood the inhibitory component of the action of ANP on pituitary-adrenal secretory activity prevails.
ENHANCED release of pituitary-adrenal hormones has been considered an essential mediating factor in the pathogenesis of various psychosomatic diseases (1, 2, 3). Therefore, an understanding of the inhibitory control of this axis in the human is of utmost clinical relevance. Recent studies have provided strong evidence for the existence of inhibitory mechanisms suppressing pituitary-adrenal responsiveness to exogenous secretagogues during early sleep (4, 5, 6). In those studies in healthy men, the ACTH/cortisol secretory response to CRH, vasopressin (VP), and combined administration of these secretagogues during early sleep was substantially lower than that during the same time of night while the men were awake. The effect of sleep could be abolished by pretreating the subjects with canreonate, presumably blocking central nervous mineralocorticoid receptors (7). Together, those studies speak for the existence of a hypothalamic release inhibiting factor of ACTH that is released, for example, during early sleep and lowers the sensitivity of this system to exogenous administration of ACTH secretagogues as well as to stress-related stimulation.
Although in humans the factors involved in the inhibitory control over pituitary-adrenal secretory activity are completely obscure, animal and in vitro studies have revealed several potential candidates for this function. Among them, atrial natriuretic peptide (ANP) was suspected to exert a suppressive effect on pituitary-adrenal activity in the human. ANP, originally isolated from the cardiac atria (8), has also been detected in the central nervous system, with particularly high concentrations in the ventral part of the periventricular nucleus of the hypothalamus (9). ANP has been shown to bind to anterior pituitary cells (10) and has been detected in hypophyseal portal blood in 3–4 times higher concentrations than those in peripheral blood (11). In vitro studies revealed an inhibitory effect of ANP on ACTH release stimulated by CRH or VP (12, 13, 14). In vivo studies in rats with intracerebroventricular administration of ANP reported an inhibition of the stimulated release of ACTH and cortisol (15) as well as a decreasing effect of the peptide on plasma VP concentrations. Studies in humans, restricted to the iv administration of ANP, have remained inconclusive about a possible role for ANP in the inhibitory control of pituitary-adrenal secretory activity. Although in a recent study, Kellner et al. (16) observed a slight reduction of CRH-induced release of ACTH and cortisol during the infusion of 150 µg ANP, Ur et al. (17) failed to disclose a clear inhibitory influence of the peptide. Part of the divergent results may derive from the fact that in humans, the effects of ANP can be determined only after iv administration. The high systemic ANP levels achieved with this route of administration may invoke peripheral effects that could mask the peptide’s action on pituitary release of ACTH.
The present experiments aimed at a closer examination of the potential inhibitory influence of ANP on the stimulated release of ACTH and cortisol in humans using different doses of ANP and different durations of ANP infusion. Also, the effects of ANP on ACTH/cortisol secretory responses to CRH and to the combined administration of CRH and VP were compared.
Subjects and Methods
Subjects and general procedure
Healthy nonsmoking men (age, 20–27 yr) participated voluntarily in the experiments after written informed consent had been obtained. Medical examination excluded any physical or psychiatric disease. Subjects were of normal weight (body mass index, 22.5 kg/m2) and did not take any medication at the time of the experiments. They had normal blood pressure and regular sleep habits. Subjects had completed their last meal (including drinks) at least 3 h before the beginning of a session, which always started at 2230 h and lasted for 2.5 h (Exp I) and 3.5 h (Exp II and III), respectively. During experimental sessions, the men rested on a bed, but stayed awake, and they were not allowed to drink or eat. Between 2230–2300 h they were prepared for blood sampling and substance administration. A forearm catheter was inserted into both antecubital veins. One was used to administer ANP (Clinalfa, Laufelfingen, Switzerland), CRH (Ferring, Kiel, Germany), and VP (Parke-Davis, Gwent, UK); the other was used for blood sampling. To keep the catheter for blood sampling patent, saline solution was infused slowly (0.3 mL/min). Blood samples were collected every 15 min starting at 2330 h to determine ANP, ACTH, cortisol, and VP. Experiments were performed in a double blind manner, and the order of treatment conditions was balanced across subjects in each experiment. The specific protocols of the three experiments are outlined below.
Exp I
In this experiment, seven healthy men (20.1–26.1 yr of age) participated, who were tested on two occasions. On one occasion, after a 15-min baseline period (2300–2345 h), 200 µg ANP were infused within 45 min (4.4 µg/min) between 2345–030 h. On the other occasion, saline solution (0.9% NaCl) was given instead of ANP. On both nights, a bolus of 50 µg CRH was injected at 2400 h, i.e. 15 min after the infusion of ANP or placebo had started.
Exp II
In this experiment, eight men (24.0–27.3 yr of age) participated, who were tested with three experimental conditions using a procedure similar to that of Exp I. The conditions were 1) ANP infused at a rate of 4.4 µg/min over 90 min, resulting in a total dose of 400 µg ANP; 2) ANP infused at a rate of 8.8 µg/min over 90 min (total amount, 800 µg ANP); and 3) a 90-min placebo infusion. Note that the infusion interval (90 min) was twice as long as that in Exp I. Again, infusion started after a baseline epoch and 15 min before the bolus injection of 50 µg CRH (injected at 2400 h). The recording epoch after CRH bolus injection was extended by 1 h (to 180 min) to enable a closer analysis of the effect of the late increase in ACTH/cortisol concentrations observed in Exp I on the ANP condition. In addition, plasma osmolality and hematocrit were measured (at the beginning and end of a session), urine was collected when subjects signaled the need, and heart rate was monitored during substance infusion.
Exp III
In this experiment the role of VP in the suppressive influence of ANP on pituitary-adrenal responsiveness should be characterized. Each of eight men was tested on two occasions after combined administration of CRH (50 µg) injected as bolus (2400 h) at 5 min of a 10-min infusion of 0.5 IU VP. Although in one condition subjects were infused over 90 min with ANP (4.4 µg/min) starting 15 min before the injection of CRH, in the other condition subjects received placebo. Otherwise the procedures and time schedule were the same as those in Exp II.
Assays and data analysis
Blood samples were immediately centrifuged, and plasma was stored at -20 C. Plasma ANP, ACTH, cortisol, and VP were all determined by RIA [ANP: Nichols Institute (Bad Nauheim, Germany); sensitivity, 15 pg/mL; inter- and intraassay coefficients of variation, respectively, <14.5% and <7.7% between 0–1000 pg/mL; ACTH: Nichols Institute; sensitivity, 1.0 pg/mL; inter- and intraassay coefficients of variation, <8% and <4%, respectively, between 1.0–1500 pg/mL; cortisol: Hermann Biermann (Bad Nauheim, Germany); sensitivity, 0.2 µg/dL; inter- and intraassay coefficients of variation, <5% and <3%, between 1.0–50 µg/dL; VP: Hermann Biermann; sensitivity, 0.6 pg/mL; inter- and intraassay coefficients of variation, <20.2% and <9.53%, respectively, between 1.2 and 80.0 pg/mL]. All samples from the same subject were determined in duplicate in the same assay.
Mean (±SEM) hormonal concentrations were determined separately for each experimental condition and for each time of blood collection. In addition, area under the response curve (AUC) measures were assessed. Statistical evaluation was based on analysis of covariance (ANCOVA) using the plasma hormone concentrations measured before substance administration as the covariate. ANCOVA included a treatment factor and a time factor, with the latter representing the time points of blood collection after substance administration. ANCOVA was also run separately for each time point of blood collection, after significance had been confirmed for a treatment effect or a treatment-time interaction effect. P < 0.05 was considered significant.
Results
Exp I
Figure 1
summarizes profiles of
plasma concentrations of ANP, ACTH, cortisol, and VP for both
experimental conditions in Exp I. Plasma concentrations of ANP were, as
expected, significantly enhanced during the 45-min ANP infusion,
reaching a maximum of 963.8 pg/mL at the end of this interval. Baseline
ANP levels were recovered within 45 min after the end of the infusion.
ANP infusion did not affect basal concentrations of ACTH and cortisol
during the 15-min interval preceding the injection of CRH.
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).
Rather than developing a clear peak, in the ANP condition, plasma
concentrations of ACTH and cortisol after CRH injection attained a
plateau at an intermediate concentration. Although ACTH concentrations
during the first 60 min after CRH injection were lower in the ANP than
in the placebo condition, during the second hour after CRH
(i.e. 60–120 min postinjection) they tended to be higher in
the ANP than in the placebo condition. Likewise, cortisol
concentrations at the end of the placebo condition fell significantly
(P < 0.05) below those in the ANP condition. Table 1 shows the AUC concentrations of ACTH
and cortisol for the first and second 60-min intervals after CRH
injection. AUC concentrations of both ACTH and cortisol were reduced by
ANP during the first hour after CRH administration. The increase in AUC
after ANP during the second 60-min interval after CRH injection reached
significance only for ACTH. The AUC for the total 0- to 120-min
interval after CRH was comparable for the ANP and placebo conditions
(ACTH, 293.20 ± 17.63 and 265.33 ± 17.71 pg/mL·min;
cortisol, 134.13 ± 9.51 and 121.08 ± 6.07 µg/dL·min for
placebo and ANP, respectively; P = NS). VP was not
influenced by ANP infusion.
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Figure 2 shows mean profiles of
plasma concentrations of ANP, ACTH, cortisol, and VP for the three
treatment conditions of Exp II. ANP infusion yielded the expected
increase in ANP concentrations; these values were about twice as high
during the infusion of ANP at a rate of 8.8 µg/min as during ANP
infusion at a rate of 4.4 µg/min. Infusion of ANP again did not
affect ACTH and cortisol plasma concentrations as well as VP
concentrations during the 15 min preceding CRH injection.
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The dynamic of the effect of ANP on stimulated ACTH/cortisol
concentrations was likewise reflected by AUC measures calculated
separately for the first, second, and third hours after CRH injection
(Table 1). Although the early suppressive effect of ANP on ACTH release
during the first hour after CRH injection was meager and remained
nonsignificant even with the low dose of ANP, the late increasing
effect of ANP during the third hour after CRH injection reached
significance at both doses of ANP. Compared with ACTH, cortisol
concentrations appeared to be more sensitive to the early suppressive
effect of ANP. Cortisol concentrations during the first hour after CRH
injection were substantially lower after ANP than after placebo, in
particular at the high 8.8 µg/min dose of ANP, but cortisol
concentrations during the third hour after CRH injection were,
conversely, higher after ANP than placebo.
VP concentrations in the placebo condition, on the average, varied between 0.6–1.0 pg/mL. During the infusion of 4.4 µg/min ANP, VP levels were even slightly lower, with this decrease reaching significance between 15–45 min after the start of the infusion. However, no decrease in VP concentrations was observed with the higher dose of 8.8 µg/min ANP. On the contrary, during the high dose of ANP, average VP plasma levels continuously increased to a maximum of 3.4 pg/mL at the end of the infusion. Yet this increase failed to reach significance, and inspection of individual data showed that it was caused mainly by two men displaying a huge rise in VP concentrations after ANP administration.
The results for VP were complemented by measures of hematocrit at the end of the sessions and of urine volume during the ANP infusion period. Although serum osmolality did not differ among the three conditions, hematocrit after the high dose of ANP (149.0 ± 3.6 g/L; P < 0.05 vs. placebo) and that after the low dose of ANP (146.4 ± 3.7 g/L; P < 0.1) were higher than that after placebo infusion (141.4 ± 5.0 g/L). Also, with high and low ANP infusions, seven and five subjects, respectively, signaled the need to urinate (yielding average volumes of 0.74 and 0.39 L, respectively), whereas this need was never signaled in the placebo condition. In parallel, heart rate averaged 74.22 ± 4.51 beats/min during the infusion of high doses of ANP and 72.19 ± 4.58 beats/min during the infusion of low doses of ANP, which was substantially higher than that during the infusion of placebo (65.0 ± 4.85 beats/min; P < 0.01 for the difference between the effects of 8.8 µg/min ANP and placebo).
Exp III
Combined administration of CRH and VP in Exp III induced the most
pronounced rises in plasma ACTH and cortisol concentrations. ANP
infusion blunted this response (Fig. 3).
ANP reduced the maximum concentration of ACTH to about one third of
that during placebo infusion (32.3 ± 5.5 vs. 86.4
± 14.5 pg/mL; P < 0.01) and the maximum cortisol
concentration to about half of that during placebo (10.7 ± 0.7
vs. 20.8 ± 0.6 µg/dL; P < 0.01).
Although ACTH concentrations were slightly elevated in the ANP session
during the third hour after CRH/VP injection, cortisol secretion
remained suppressed after ANP throughout the 3-h recording epoch after
CRH/VP administration. Consequently, AUC measures revealed the
suppression of the ACTH secretory response by infusion of ANP to be
significant for the first and second hours after CRH/VP administration,
and the suppression of cortisol secretion to be significant for the
first, second, and even third hours after CRH/VP administration (Table 1).
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The experiments investigated the effects of ANP infusion on the ACTH/cortisol secretory response to CRH and combined administration of CRH and VP in healthy men. Results indicate an early suppressive effect of ANP on pituitary-adrenal responsiveness developing within 60 min after administration of the ACTH secretagogues. Thereafter, suppressive influences become replaced by an enhancing influence of ANP on ACTH/cortisol release, which was most pronounced during the third hour after administration of the secretagogues. The early suppressive effect of ANP infusion was more pronounced on concentrations of cortisol than ACTH, and was also more pronounced after combined administration of CRH and VP than after sole injection of CRH. The early suppression may reflect a direct inhibitory action of ANP on both adrenal release of cortisol and pituitary release of ACTH. In contrast, the late increase in ACTH and cortisol appears to reflect a response to secondary release of ACTH secretagogues contingent upon enhanced diuresis.
The present findings are in line with previous studies in humans indicating that ANP can reduce pituitary-adrenal responsiveness to its secretagogues without affecting basal concentrations of ACTH/cortisol (16, 18). Yet, in those studies the suppressive actions of ANP (infused at a rate of 5 µg/min over 30 min) were of moderate size. In another study (17), ANP infused at an even lower rate (0.01 µg/kg·min over 180 min) completely failed to affect ACTH/cortisol secretory responses to CRH. This led us to examine the effects of statistically higher doses of ANP, and the duration of ANP infusion was varied. At a dose of 4.4 µg/min infused over 90 min, in fact, a clear immediate suppressive action of ANP on ACTH/cortisol secretory responses was revealed. However with a further increased rate of infusion (to 8.8 µg/min over 90 min), the immediate inhibitory action of ANP became smaller again, probably due to effects secondary to the diuretic action of ANP.
This view derives from the results in rats reported by Kovacs and Antoni (15), which bear some similarities with the present pattern of changes. Those authors examined concentrations of ACTH after systemic administration of ANP and CRH/VP in rats with lesions of the nucleus paraventricularis of the hypothalamus and in sham-operated controls. In controls, as in the present sample of healthy humans, low doses of ANP reduced stimulated release of ACTH, associated with a transient hypotensive effect. In contrast, high doses of ANP inducing a distinct and long lasting hypotension, failed to inhibit stimulated ACTH release. Notably, in the lesioned rats with nearly 90% of hypophysiotropic cells eliminated, high doses of ANP blunted ACTH secretory responses to CRH/VP despite the presence of substantial hypotensive effects. Based on these results, the researchers proposed that ANP inhibits CRH/VP-stimulated ACTH secretion at the pituitary level, but this effect may be masked at a high dose of ANP, provoking a stimulatory effect on the hypophysiotropic cells of the paraventricularis of the hypothalamus that may override the inhibiting actions of the peptide.
In fact, it is well documented that hypovolemia with consecutive hypotension, as observed during ANP, represents a condition stimulating CRH/VP release (19, 20, 21). Several indexes suggest that similar conditions exist in the present sample of human subjects during infusion of the high dose of ANP. Hematocrit was reduced and collected urine enhanced in this condition. Parallel increases in plasma concentrations of VP and in heart rate suggest a compensatory response to hypovolemia. The emergence of hypotension would have further supported this view. However, blood pressure was not monitored continuously in this study. Interestingly, Bähr et al. (21) provided evidence that thirst-induced VP release in men (presumably from the neurohypophysis) is accompanied by an enhanced secretory activity of the pituitary-adrenal axis. Together with those findings, the present changes after ANP treatment suggest that hypovolemic conditions could have been present with high doses and also with prolonged infusion of the peptide, which, in turn, exerted a stimulating effect on the release of hypothalamic secretagogues of ACTH. Considering that in portal blood endogenous release of CRH/VP can lead to concentrations 10- to 100-fold higher than those in systemic blood (22, 23), it is conceivable that endogenous release of CRH/VP overrode any inhibitory effect of exogenous ANP. Apart from its effects on the pituitary, ANP may in addition directly inhibit cortisol release at the adrenal level, as the early inhibitory effect of ANP appeared to be more pronounced on secretory responses of cortisol than on those of ACTH (24, 25).
As expected, combined CRH/VP stimulation potentiated ACTH/cortisol secretory responses in the placebo condition (26, 27). The immediate inhibitory effect of ANP on ACTH/cortisol release after combined CRH/VP administration was distinctly stronger than that after sole administration of CRH. Moreover, the late stimulatory effect of ANP was marginal on the release of ACTH and totally absent on cortisol release. Two-fold actions of VP may have contributed to this pattern. First, the vasoconstrictor activity of VP could have compensated for hypovolemic and hypotensive actions of ANP. Secondly, exogenous VP could have temporarily suppressed endogenous release of VP from hypophysiotropic cells of the hypothalamus (28), thus preventing a pronounced activation of mechanisms counteracting the hypovolemic actions of ANP. This view was supported by a post-hoc inspection of data from Exp II, which revealed that the inhibitory effect of the high doses of ANP became more pronounced when data from two subjects with extraordinarily high increases in plasma VP concentrations (presumably indicating strong responses to hypovolemia) were eliminated.
The pattern of changes observed here indicates an inhibitory effect of exogenous ANP on pituitary-adrenal responsiveness to CRH and combined CRH/VP. However, with prolonged infusion and at high doses of ANP, this effect becomes overridden by a stimulating influence of the peptide on ACTH/cortisol release. The inhibitory effect of ANP on pituitary ACTH release could be based on the well known antagonism between cGMP mediating the cellular response to ANP, and cAMP and calcium as second messengers in the response to CRH/VP (13, 29). The fact that the inhibitory influence of ANP on pituitary-adrenal responsiveness is increasingly obscured by stimulation of ACTH/cortisol release most likely originates from peripheral diuretic effects of the peptide after exogenous administration. If this is the case, endogenous release of ANP into portal hypophysial blood would be expected to yield an inhibition of pituitary-adrenal secretory activity much more efficient than that induced experimentally after systemic administration of ANP. However, to conclude from the present results that ANP plays a significant role in the inhibitory control of the pituitary-adrenal system would be clearly premature as long as actual concentrations of the hormone in portal blood are unknown and its relation to C-type natriuretic peptide is obscure (this is another member of the natriuretic peptide family that appears to be highly important in this context) (30).
Acknowledgments
The authors thank A. Otterbein, S. Baxmann, and C. Zinke for excellent technical assistance.
Received July 1, 1997.
Revised December 5, 1997.
Accepted December 22, 1997.
References
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