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Departments of Neuroendocrinology (B.P., B.S., C.D., J.B., H.L.F.) and Internal Medicine I (B.P., B.S., B.B., C.D., H.L.F.), University of Lübeck, D-23558 Lübeck, Germany
Address all correspondence and requests for reprints to: Boris Perras, M.D., Medizinische Klinik I, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, D-23558 Lübeck, Germany. E-mail: Perras{at}kfg.mu-luebeck.de.
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Abstract |
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Thirty minutes after intranasal administration of ANP (1 mg) and placebo, pituitary-adrenal activity was stimulated in 18 healthy men by two tests: 1) a standard insulin-hypoglycemia test and 2) CRH combined with vasopressin (VP), respectively. ACTH, cortisol, VP, blood pressure, heart rate, and measures of fluid balance were also recorded.
Pretreatment with ANP suppressed cortisol (P < 0.01) and ACTH (P < 0.05) secretory responses to insulin-induced hypoglycemia to about half of that seen after placebo, but pituitary-adrenal activity was not suppressed by CRH/VP injection (P > 0.7). Indicators of fluid balance, cardiovascular parameters, and self-report measures were not influenced by the treatment.
Results indicate a strong inhibition of stimulated pituitary-adrenal activity after intranasal administration of ANP. The absence of an effect on CRH/VP-induced pituitary-adrenal responses suggests a direct action of the peptide on the central nervous system inhibiting stimulated hypothalamo-pituitary-adrenal activity at the hypothalamic level.
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Introduction |
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In recent years, it has become evident that overactivity of the HPA axis is not only characteristic of rare illness like Cushing’s disease but is a frequent symptom in widespread diseases like diabetes mellitus, obesity, hypertension, depression, and other psychiatric disorders, where it contributes to the development and manifestation of these conditions (5, 6, 7, 8, 9, 10). In the elderly, hypercortisolism is considered to enhance the physiological sequelae of aging (11, 12, 13). It has been hypothesized recently that insufficient inhibition of the pituitary-adrenal system with altered feedback is a consequence of increased cumulative load of lifetime stress (allostatic load) (9, 11, 12). Although of utmost clinical relevance, knowledge about the inhibitory control over the human HPA system beyond the negative feedback regulation by glucocorticoids is still scarce. On this background, a means to improve inhibition of HPA activity would not only help to clarify basic mechanisms of HPA regulation but also would help to understand the resiliency of the HPA system in various diseases and, eventually, to treat hyperactivity of this axis.
Here, we examined effects of intranasal ANP on stimulated secretion of cortisol and ACTH to test the hypothesis that ANP inhibits pituitary-adrenal activity at a central nervous level. For administration of ANP, the nose-brain pathway was chosen to enable the peptide to access the brain compartment directly and to avoid confounding effects by systemic actions of ANP (14). In animals, histochemical studies have demonstrated that proteins and peptides can enter easily the brain via the nose-brain pathway (15, 16, 17, 18, 19). Determining different model peptides like vasopressin (VP), ACTH, and insulin in the cerebrospinal fluid, previous experiments in humans have shown that via nasal administration neuropeptides directly enter the cerebrospinal fluid compartment (20, 21). The view of a direct passage of peptides to the brain was consistently further confirmed by experiments in humans providing functional evidence for a direct passage of substances to the brain. Using evoked potentials and other neurophysiological methods brain function was influenced while the amount of substance simultaneously entering the blood stream after nasal administration was in most of these cases negligible (22, 23, 24, 25, 26). Thus, we expected that ANP after intranasal administration induces central nervous actions without interfering influences on body fluid homeostasis due to uptake into the circulation (27). Any possible influences of the procedure of nasal substance administration were controlled by the placebo condition in which saline solution was administered intranasally. Furthermore, ANP vs. placebo conditions were compared in a double-blind manner. To discriminate effects of ANP on the pituitary from effects on suprapituitary brain regions, two different stimulatory procedures were compared: the CRH/VP test stimulates secretion of ACTH and cortisol at the pituitary level (28), whereas the insulin-hypoglycemia (ins/hypo) test stimulates pituitary-adrenal activity via glucose sensors located in the hypothalamus (29).
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Subjects and Methods |
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Eighteen healthy male students were tested. They were normal-weight nonsmokers and had no history of cardiovascular, metabolic, or psychiatric disease. They did not drink alcohol or coffee and were free of any medication during the period of the experiments. They were not working on night shifts and had not eaten for at least 6 h before the experimental sessions. Mean age was 27.3 yr (range, 25–30 yr). Subjects were paid and had given informed consent before examination. The study was approved by the local ethics committee.
Procedure and design
Each of the 18 men was examined on two occasions in a double-blind, placebo-controlled crossover study design. The subject’s two experimental sessions were separated by at least 1 wk. Sessions started at 1200 h. Intravenous catheters were inserted in an antecubital vein of each arm. One served for blood sampling, the other for substance administration. Then, the pretreatment of 1 mg ANP or placebo was administered intranasally in randomized order. Human ANP (hANP1–28) was obtained from Calbiochem (Bad Soden, Germany). For intranasal administration, 1 mg ANP was diluted in 3.5 ml saline solution and administered by a nasal spray atomizer every 30 sec in both nostrils over 10 min. Saline solution served as placebo.
Subsequently, subjects were seated in a reclining chair and were allowed to read a magazine. Twenty minutes later, nine men were subject to a modified standard ins/hypo test and the other nine men to a CRH/VP test. For the ins/hypo test, 0.12 IU rapid-acting regular insulin per kilogram of body weight (Aventis, Frankfurt, Germany) was injected as bolus. Blood glucose was monitored closely in 5-min intervals (HemoCue B-glucose analyzer, HemoCue GmbH, Großostheim, Germany), and hypoglycemia was stopped by infusion of a 10% glucose solution when glucose concentration reached 40 mg/dl. Our aim was to prevent severe neuroglycopenic symptoms and to avoid any risk of seizures. Normally, the counterregulatory response of the HPA axis and of other hormones is initiated when blood glucose concentration falls less than 50 mg/dl, although with great individual differences (30). Three subjects of this group were replaced because they failed to respond with any increase in ACTH and cortisol concentration on the placebo condition, despite blood glucose nadir values less than 40 mg/dl. On the CRH/VP test, 0.5 IU arginine-VP (Parke-Davis, Karlsruhe, Germany) diluted in 100 ml saline solution was infused over 6 min. In the 3rd minute, 50 µg CRH (Ferring, Kiel, Germany) was injected. To determine plasma concentrations of ACTH, cortisol, VP, serum sodium concentrations, and hematocrit, blood was sampled during a period ranging from 30 min before and 150 min after the stimulation tests. To determine ANP plasma concentrations, additional blood was collected every 5 min between 0 and 35 min post-ANP and placebo pretreatment and subsequently every 15 min for another 90 min. Blood pressure and heart rate were measured automatically (Bosch und Sohn, Jungingen, Germany) in parallel with the sampling of blood for determining hormone concentrations. Urine for assessment of volume and sodium concentration was collected once at the end of the total 2.5-h experimental interval after subjects had emptied their bladder at the beginning of the experiment. At the beginning of the session, immediately before the stimulation tests, 30 and 60 min after, as well as at the end of each session subjects reported their feeling of hunger, thirst, and vigilance. This was done by self-ratings on 11-point scales ranging from 0 (feeling satiated, not thirsty, and highly vigilant, respectively) to 10 (feeling very hungry, very thirsty, and very drowsy, respectively).
Assays and statistical analysis
Blood samples were centrifugated, and plasma was immediately stored at –20 C for later determination of hormone concentrations. The following commercially available assays were used: cortisol, RIA [DPC Biermann, Bad Nauheim, Germany; sensitivity, 0.2 µg/dl (5.52 mmol/liter), intraassay coefficient of variation (CV) < 5.1% between 1 and 50 µg/dl (27.6 and 1379.5 mmol/liter, respectively)]; ACTH, immunoluminometric assay [Brahms, Hennigsdorf, Germany; sensitivity, 1.0 pg/ml (0.22 pmol/liter), intraassay CV < 4.9% between 4.2 and 347 pg/ml (0.92 and 82.28 mmol/liter, respectively)]; ANP, RIA [Euro-Diagnostica, Arnhem, The Netherlands; sensitivity, 3.5 pg/ml (1.14 pmol/liter), intraassay CV < 8.6% between 3.5 and 1000 pg/ml (1.14 and 326 pmol/liter, respectively)]; and VP, RIA after extraction [Bühlmann Laboratories, Allschwill, Switzerland; sensitivity, 1.25 pg/ml (1.16 pmol/liter), intraassay CV < 11.2% between 1.7 and 20.0 pg/ml (1.58 and 18.6 pmol/liter, respectively)]. Interassay CV was for all assays less than 12%. All samples from an individual were measured in duplicate within the same assay.
Statistical analysis was based on analyses of covariance, including a repeated measures factor for treatment (ANP, placebo) and a time factor (representing the multiple measurements after the stimulation tests). The measurement immediately before the stimulation tests served as covariate. Results after the ins/hypo and CRH/VP tests were analyzed separately. Results from the self-report questionnaire were evaluated by nonparametric tests (Wilcoxon, Kruskal-Wallis). P < 0.05 was considered significant.
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Results |
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ANP plasma levels (illustrated in Fig. 1
), in general, remained at the lower end of the normal assay range throughout the study [9–68 pg/ml (2.9–22.2 pmol/liter)]. Compared with placebo, ANP plasma concentrations were slightly increased 25–30 min after the beginning of intranasal ANP administration [mean ± SEM peak ANP plasma concentration, 21.5 ± 1.4 pg/ml (7.0 ± 0.3 pmol/liter); placebo, 15.6 ± 1.4 pg/ml (5.1 ± 0.3 pmol/liter); P < 0.01], indicating that a minor amount of peptide was resorbed into the circulation. However, the increase was only transient, and baseline concentrations were fully recovered before the stimulation tests. Thereafter, ANP plasma concentrations remained comparable in both treatment groups and were not influenced by the stimulation tests. There were also no effects of intranasal ANP on basal plasma concentrations of cortisol and ACTH immediately after substance administration (Figs. 2 and 3).
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Blood glucose level during baseline was normal in both treatment conditions [mean ± SEM blood glucose ANP, 89.1 ± 5.6 mg/dl (4.95 ± 0.31 mmol/liter); placebo, 95.4 ± 4.4 mg/dl (5.29 ± 0.24 mmol/liter); P > 0.9]. After insulin injection, blood glucose rapidly fell to nadir values, which also were closely comparable in both conditions [ANP, 39.9 ± 3.5 mg/dl (2.21 ± 0.19 mmol/liter); placebo, 37.2 ± 3.7 mg/dl (2.06 ± 0.21 mmol/liter); P > 0.6]. Although the hypoglycemia induced in our experiments was moderate, it should be emphasized that it was clearly comparable regarding the average value and the variance between both experimental conditions.
Pretreatment with ANP inhibited stimulated secretion of ACTH (Fig. 2
, top) between 45 and 75 min after hypoglycemia so that peak plasma concentrations were only half of that seen in the placebo condition [22.4 ± 7.6 vs. 52.3 ± 8.1 ng/ml (4.9 ± 1.7 vs. 11.5 ± 1.8 pmol/liter); P < 0.05]. Calculation of area under the curve (AUC; baseline referenced) revealed a distinctly smaller AUC after ANP [12,106.5 ± 1974 ng/ml·min (2663.4 ± 434.3 mmol/liter·min)] than after placebo [21,777.0 ± 1974 ng/ml·min (4790.9 ± 434.3 mmol/liter·min); P < 0.05]. Cortisol secretion was suppressed to a similar extent between 60 and 90 min after insulin injection (Fig. 2, bottom). Thus, plasma peak concentration after ANP averaged only 12.9 ± 1.6 µg/dl (356.0 ± 44.2 mmol/liter) but amounted to 19.3 ± 1.7 µg/dl (532.7 ± 46.9 mmol/liter) after placebo (P < 0.01). Cortisol AUC after ANP averaged 7812.0 ± 861 µg/dl·min [215.6 ± 23.8 mol/liter·min and after placebo 11,508.0 ± 861 µg/dl·min (317.6 ± 23.8 mol/liter·min); P < 0.01].
Effects of ANP on pituitary-adrenal responses to the CRH/VP test
The combined administration of CRH and VP induced a strong rise in plasma ACTH and cortisol concentration. However, in contrast to the responses to the ins/hypo test, these responses did not differ between ANP and placebo pretreatment conditions (Fig. 3). Peak concentrations and time courses of ACTH and cortisol concentrations after CRH/VP testing were almost identical for the ANP and placebo conditions [ACTH peak concentration after ANP, 67.8 ± 9.1 ng/ml (14.9 ± 2.0 pmol/liter); placebo, 67.2 ± 10.0 ng/ml (14.8 ± 2.2 pmol/liter); P > 0.7; cortisol peak concentration after ANP, 18.4 ± 1.1 µg/dl (507.8 ± 30.4 mmol/liter); placebo, 17.8 ± 1.3 µg/dl (491.3 ± 35.9 mmol/liter); P > 0.7].
Fluid balance, heart rate, and blood pressure
Repeated measurements of hematocrit and concentrations of serum sodium and plasma VP as well as assessment of urine output and urine sodium did not indicate any detectable changes in these indicators of fluid balance after intranasal ANP (Table 1).
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). Self-reported feelings of hunger, thirst, and vigilance
Questionnaire data revealed that at the beginning of a session subjects felt slightly hungry [median score ANP, 3.5 (1–7); placebo, 3.5 (0–7)] and thirsty [median score ANP, 4.0 (2–7); placebo, 3.5 (0–6)], and also vigilance was at an intermediate level [median score ANP, 4.5 (2–7); placebo, 2.0 (0–6)]. In the course of the experiment, no treatment-associated effects were detected, and at the end of the session, ratings of hunger [median score ANP, 5.5 (2–9); placebo, 6.0 (3–9)], thirst [median score ANP, 5.5 (3–9); placebo, 6.5 (2–8)], and vigilance [median score ANP: 3.5 (2–8); placebo: 2.5 (0–8)] were also well comparable between the treatment conditions (P > 0.1 for all tests).
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Discussion |
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In animals, ANP has been shown to inhibit pituitary-adrenal activity after intracerebroventricular administration (2). In human studies employing the intravenous route of administration, conflicting results have been revealed with both inhibitory and lacking effects after ANP (31). The discrepancies among human experiments may be explained by different ANP doses with only higher intravenous doses reaching the brain in sufficient amounts (27, 32, 33, 34). Convergent support for a brain-mediated inhibition of the HPA system derives from in vitro and animal studies that have revealed ANP immunoreactive neurons and a high density of ANP receptors in brain areas like the preoptic and periventricular nuclei of the hypothalamus (2) as well as an inhibition of stress-induced release of CRH, VP, and ACTH after intracerebroventricular injections of ANP (35, 36, 37, 38, 39, 40). After systemic administration or in cultured pituitary tissue, ANP failed to affect induced ACTH secretion (41). These findings do not rule out any additional influence of bloodborne ANP on pituitary function. The pituitary per se expresses ANP receptors and may also respond to ANP with reduced synthesis of proopiomelanocortin peptides (42, 43, 44). Overall, however, these data provide little support for the view that systemic ANP by a direct action on the pituitary could exert an inhibitory control over ACTH/cortisol release.
Importantly, here we found no substantial increase in plasma ANP concentration after intranasal substance administration. Only a marginal fraction of the peptide was resorbed into the blood stream as indicated by a slight elevation of ANP plasma concentrations 25–30 min after the beginning of the intranasal administration. During the period of stimulation tests and in the further course of the experiment, ANP plasma concentrations were closely comparable in both treatment conditions and remained at the lower end of the normal range. Considering also previous human studies using intravenous ANP administration, where an inhibition of pituitary-adrenal release was found only with up to 200-fold increases of blood ANP concentrations, any contribution of circulating ANP to the effects observed here in conjunction with insulin-hypoglycemia-induced responses can be safely ruled out (27).
The findings that blood pressure and markers of fluid balance (serum sodium, hematocrit, urine sodium, urine volume) were not influenced by ANP treatment likewise corroborates the view that peripheral target organs of ANP (heart, vasculature, and kidneys) were not reached by the peptide (1). On the other hand, after intracerebroventricular administration of ANP in animals, a decrease in blood pressure-enhanced electrolyte excretion and diuresis and suppressed VP secretion and drinking behavior were also found (for review, see Ref. 2). That our results did not reveal similar effects after intranasal administration is difficult to integrate. The induction of these effects may require substantially higher ANP concentrations in the cerebrospinal fluid and in relevant brain regions than achieved here by the nasal route of administration in humans. Also, our experimental design was not particularly sensitive to changes in fluid balance. Insulin-induced hypoglycemia is a well-known and strong stimulus of thirst that may have masked weaker influences of ANP.
We conclude that ANP via the nose-brain pathway reached the brain directly to inhibit hypothalamic release of ACTH secretagogues after hypoglycemia. For several neuropeptides like VP, insulin, MSH/ACTH4–10, cholecystokinin, and growth hormone releasing-hormone, evidence has been provided that they reach the human brain compartment after nasal administration directly (26, 45). The amount of intranasal ANP that reached the circulation was very small (as estimated by the change in plasma ANP concentrations) and would not have enabled passage of the substance to the brain in sufficient quantity because ANP does not easily penetrate the blood-brain barrier (46, 47). Our findings of a central nervous action of ANP support the idea, that ANP acts as a corticotropin release-inhibiting factor and, according to a recently proposed hypothesis (4), belongs to a probably larger group of brain-derived neuromodulators of pituitary-adrenal regulation. ANP may play an important role in the brain’s repertoire to inhibit stress-related activation of the HPA system in humans (48), although besides ANP, several other candidates, like a TRH prohormone fragment, somatostatin, dopamine, etc., have been considered as possible inhibitors of HPA activity (3, 4). Considering also that the nasal route of substance administration used here proved practicable and was free of side effects, our data suggest intranasal ANP as a possible therapeutic approach to diseases characterized by hyperactivity of the pituitary-adrenal system.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANP, Atrial natriuretic peptide; AUC, area under the curve; CV, coefficient(s) of variation; HPA, hypothalamo-pituitary-adrenal; ins/hypo, insulin-hypoglycemia; VP, vasopressin.
Received January 19, 2004.
Accepted May 26, 2004.
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