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Divisions of Cardiology (D.D.S.), Endocrinology and Metabolism (D.L.V., G.I.S.M., I.H.), Departments of Internal Medicine, Physiology and Biophysics, University of South Florida Health Sciences Center and James A. Haley Veterans Medical Center, Tampa, Florida 33612
Address all correspondence and requests for reprints to: David L. Vesely, M.D., Ph.D., Director, Atrial Natriuretic Peptides Research Laboratories, James A. Haley Veterans Hospital, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: vesely.david_l{at}tampa.va.gov
Abstract
The present investigation was designed to determine whether atrial natriuretic peptides consisting of amino acids 1–30 (i.e. long-acting natriuretic hormone), 31–67 (vessel dilator), 79–98 (kaliuretic hormone), and 99–126 (atrial natriuretic hormone) of the 126 amino acid atrial natriuretic hormone prohormone decrease CRH, ACTH, and/or cortisol in healthy humans (n = 30). Vessel dilator, kaliuretic hormone, long-acting natriuretic hormone, and atrial natriuretic hormone decreased the circulating concentration of CRH 84%, 74%, 67%, and 62% (P < 0.001 for each), respectively, when infused at 100 ng/kg body weight·min for 60 min. Vessel dilator, kaliuretic hormone, long-acting natriuretic hormone, and atrial natriuretic hormone decreased circulating ACTH concentrations 58%, 80%, 81%, and 70% (P < 0.001) and the circulating concentration of cortisol 73%, 72%, 73%, and 67% (P < 0.001), respectively. The decreases in CRH, ACTH, and cortisol lasted 11/2 to 3 h after cessation of the respective atrial natriuretic peptide infusions. These data, along with the knowledge that cortisol upregulates atrial natriuretic peptides’ gene expression and CRH and ACTH stimulate atrial natriuretic peptides’ release, suggest that these four atrial natriuretic peptides may be part of an intricate feedback system to help regulate cortisol concentrations via their ability to decrease the circulating concentration of CRH which, in turn, results in a decrease in ACTH and cortisol.
ATRIAL NATRIURETIC HORMONE (ANH) inhibits the release of CRH in vitro (1, 2) and possibly in vivo (3). ANH inhibits basal and ACTH release in vitro (4, 5, 6). ANH also inhibits CRH-stimulated ACTH release in humans (7) and unstimulated ACTH levels in rats (8). ANH, further, has been reported to inhibit basal and ACTH-stimulated cortisol release from human adrenal tissue (9, 10, 11) and to decrease basal ACTH and cortisol levels (12). When one blocks the effects of ANH by using antisera to ANH, this results in a markedly enhanced stress-induced secretion of ACTH (13, 14) and CRH (15), suggesting that ANH is a physiologically relevant CRH-inhibiting hormone (13, 14, 15). Thus, when the inhibitory effects of ANH are no longer present after being blocked by a specific antibody, CRH increases in the circulation (15).
ANH is part of a peptide hormonal system consisting of a 126-amino acid (a.a.) prohormone synthesized mainly within myocytes of the heart and stored in storage granules within the heart for release into the circulation (16). This hormonal system is also present in several other tissues, including the hypothalamus, which contains the highest concentration of ANH prohormone mRNA within the brain (17). This hormonal system contains several peptide hormones from the same 126-a.a. prohormone with blood pressure–lowering, natriuretic, diuretic, and/or kaliuretic (i.e. potassium-excreting) properties (18, 19, 20, 21, 22). Thus, peptides consisting of a.a. 1–30 (pro-ANH 1–30; long-acting natriuretic hormone), a.a. 31–67 (pro-ANH 31–67; vessel dilator), a.a. 79–98 (pro-ANH 79–98; kaliuretic hormone), and a.a. 99–126 (pro-ANH 99–126; ANH) each have blood pressure-lowering, diuretic, natriuretic, and/or kaliuretic properties in both humans (20, 21, 22) and animals (18, 19).
Long-acting natriuretic hormone (LANH), vessel dilator, and kaliuretic hormone, which are released simultaneously with ANH (23) and have markedly similar biologic effects (16, 17, 18, 19, 20), have never been investigated as to their ability to inhibit the CRF-ACTH-cortisol axis. The present investigation was designed to determine whether infusion of vessel dilator, LANH, and kaliuretic hormone decrease the circulating concentration(s) of cortisol, ACTH, and/or CRH. In addition, ANH infusions were performed to be able to compare any findings in the present investigation with LANH, vessel dilator, and/or kaliuretic hormone with previous investigations of ANH with respect to modulation of the hypothalamic-pituitary adrenal axis.
Subjects and Methods
Experimental subjects
Thirty healthy subjects (15 men and 15 women; aged 20–58 yr; average age 32 yr; all normotensive with blood pressures < 125/80 mm Hg) were studied. These subjects had heart rates ranging from 56 to 80 beats/min, with respiration rates between 12 and 14/min. These 30 volunteers were divided into five similar groups with six individuals in each group; the age, gender, weight, blood pressure, and heart rate were as follows for each group: The means ± SEM for the healthy controls were 34 ± 4 yr, 76 ± 6 kg, 110 ± 4/71 ± 4 mm Hg, and 68 ± 2 beats/min; the LANH group averages were 31 ± 4 yr, 76 ± 6 kg, 103 ± 3/66 ± 2 mm Hg, and 68 ± 3 beats/min; the vessel dilator subjects’ means were 35 ± 5 yr, 75 ± 5 kg, 114 ± 5/72 ± 3 mm Hg, and 64 ± 2 beats/min; the kaliuretic hormone group averaged 32 ± 5 yr, 76 ± 6 kg, 108 ± 5/72 ± 5 mm Hg, and 66 ± 3 beats/min; and the ANH group means were 33 ± 6 yr, 76 ± 4 kg, 109 ± 4/75 ± 2 mm Hg, and 70 ± 4 beats/min. These parameters were not statistically different among groups. None of the volunteers had any known disease. Of importance, none of the subjects had any abnormality of sodium or water metabolism. None of the volunteers was taking any medication. Written informed consent was obtained from each of the volunteers after the nature and possible consequences of the studies were fully explained. This study was approved by the Institutional Review Board of the University of South Florida Health Sciences Center and the Research Committee of the James A. Haley Veterans Hospital and followed the guidelines of the Declaration of Helsinki. This study was also approved by the U.S. Food and Drug Administration (FDA IND 32,119). These same healthy subjects have participated previously in a study of the natriuretic, diuretic, and blood pressure–lowering effects of these peptide hormones (21).
Experimental protocol
After obtaining written informed consent, an Insyte-w 20-gauge, 1.5-in catheter was placed into the forearm of each subject for infusion and blood sampling. A 60-min baseline period preceded any infusion. A total volume of 20 ml normal saline (0.9% sodium chloride, with or without peptides) was infused by a constant rate infusion pump over a 60-min period. Blood and urine samples were obtained every 20 min during the infusion and at 30-min intervals during the 1-hr baseline and 3-hr postinfusion periods. Thus, one group (control group or sham infusion) received only 20 ml normal saline without any of the peptide hormones, and the other four groups received one of the respective peptide hormones in 20 ml normal saline. One hundred nanograms per kg BW/min was chosen for the infusion of these atrial natriuretic peptides because the rate of release of the N-terminal ANH prohormone peptides from the atrium of the heart with physiological stimuli is 138–292 ng/kg BW·min, whereas the release rate of ANH from the atrium is 76 ng/kg BW·min (24). All subjects were studied in the morning after an overnight fast, beginning their baseline period at 0800 h. Each volunteer was studied in the seated position and received only one peptide hormone infusion. Molar equivalents of the 100 ng/kg BW dose were 32, 29, 26, and 46 pmol/L·kg BW for ANH, LANH, vessel dilator, and kaliuretic hormone, respectively. Thus, the concentrations used in this investigation are in the physiological range based on the release rates for the N-terminal ANH prohormone peptides and slightly above the physiological range for ANH.
Purity of the ANHs
The human forms of LANH, vessel dilator, kaliuretic hormone, and ANH were synthesized by Peninsula Laboratories, Inc. (Belmont, CA). Before their use in these studies, samples of these commercially synthesized peptides were subjected to HPLC to determine their purity using a Novapak C18 (5-µm) cartridge column (Waters Chromatography Division, Millipore Corp., Milford, MA). The flow rate for the HPLC study was 1 ml/min with 0.1% trifluoroacetate solvent in pump A and 60% acetonitrile in 0.1% trifluoroacetate in pump B, with a gradient of 0–60% acetonitrile achieved in 40 min. This evaluation verified their purity and authenticity, compared with the known HPLC elution profile (25) of these peptide hormones. After determining that the respective peptide hormones were pure, the peptide hormones were dissolved in 0.9% saline solution in the hospital pharmacy at which pyrogen and sterility testing was performed before dispensing the 100 ng/kg BW concentrations of each peptide hormone into two 10-ml syringes. Each 10-ml syringe infused all of its content over a 30-min time period. After completing the experiment, each of the syringes and the infusion catheter were examined by their specific RIAs to determine the amount of the respective peptide hormone that may have remained within the syringes or tubing. Approximately 5% of each peptide hormone remained on the walls of the syringes and tubing after completion of the infusion. This was determined after completion of the experiment by flushing the syringes three times with 4 ml of 0.9% saline and then measuring by the respective RIAs how much of each peptide was present in the 0.9% saline flush. The amount measured was then compared with the amount infused to determine the percentage that had remained on the walls of the syringes and tubing.
Measurement of CRH
Each of the blood samples for measurement of the peptide hormones were collected into chilled 5 ml EDTA tubes to prevent proteolytic breakdown of any peptides such as CRH or ACTH that might be present. These samples were transported on ice and immediately centrifuged at 3000x g for 15 min. After centrifugation, each sample was extracted with 100% ethanol (1:1 dilution), vortexed, and allowed to stand at 4 C for 30 min. CRH was measured by RIA using 125I-labeled CRH devised to the human sequence of CRH (Phoenix Pharmaceuticals, Inc., Belmont, CA). CRH was measured in plasma samples at 0, 30, 60 (beginning of respective peptide hormone infusions), 80, 100, 120 (end of peptide infusions), 150, 180, 210, 240, 270, and 300 min in each subject (n = 30). The CRH antiserum has a sensitivity of 10.34 pg/tube and the following cross-reactivities: human CRF, 100%; ACTH, 0%; LHRH, 0%; urocortin, human and rat, 0%; brain natriuretic peptide (BNP), 0%; and pituitary adenylate cyclase activating peptide-38, 0%. The intra- and interassay coefficient(s) of variation were 6.9% and 9.7%, respectively.
ACTH measurement
ACTH was measured from the same plasma samples at each of the
above time periods from 0 to 300 min using
125I-labeled human ACTH RIA (Phoenix Pharmaceuticals, Inc., Belmont, CA). The plasma samples for this
assay were processed identically to the CRH assay above. The
IC50 for this ACTH assay is 58–113 pg/tube, and
it has the following cross-reactivities: human ACTH, 100%; rat ACTH,
100%; human ACTH 1–24, 100%; human ACTH 7–38, 31%; B-endorphin,
1%; CRF, 0%; ANH, 0%; BNP, 0%; 
-MSH, 0% and
met-enkephalin, 0%. The intra- and interassay coefficient(s) of
variation were 7.3 and 8.9%, respectively.
Serum cortisol
Serum cortisol was measured at each of the above time points (0 to 300 min) using a RIA from Diagnostic Products Corp. (Los Angeles, CA). Serum was drawn at the same time points as the plasma samples above. In this assay, serum 125I cortisol is measured directly without extraction or predilution (26). There is no centrifugation step. In this solid-phase RIA, polypropylene tubes with antibodies to cortisol (Coat-A-Count) are used at which 125I-labeled cortisol competes for a fixed time within the respective healthy volunteer sample for antibody sites. The detection limit of this assay is approximately 0.2 µg/dl. This assay is highly specific for cortisol with the following cross-reactivities: aldosterone, 0.03%; corticosterone, 0.94%; cortisone, 0.98%; danazol, 0.01%; 11-deoxycorticosterone, 0.26%; 11-deoxycortisol, 11.4%; dexamethasone, 0.04%; estriol, 0.01%; estrone, 0.007%; flumethasone, 0.017%; methotrexate, 0.004%; prednisolone, 76%; prednisone, 2.3%; pregnenolone, 0.02%; pregnanediol, 0%; progesterone, 0.02%; and tetrahydrocortisol, 0.34%. The intra- and interassay coefficient(s) of variation for cortisol were 4.7% and 5.2%, respectively.
Statistical analysis
The data obtained in this investigation are illustrated as mean ± SE. Differences in CRH, ACTH, and cortisol measurements among subjects or groups of subjects were evaluated by the one-way ANOVA. Measurements of CRH, ACTH, and cortisol obtained in the same subjects over time were evaluated by the repeated measures of ANOVA. To be considered statistically significant, we required a P value to less than 0.05 (95% confidence).
Results
LANH and ANH decreased the circulating concentration of CRH 67%
and 62% (P < 0.001), respectively, during their
infusions in healthy humans (Fig. 1
). The
maximal decrease (i.e. 75%, P < 0.001) in
CRH secondary to ANH occurred 90 min after its infusion ceased (Fig. 1). The circulating concentration of CRH returned to baseline 2 h
after ANH infusion and remained at its baseline concentrations
throughout the rest of the investigation (Fig. 1). On the other hand,
CRH was 67% and 45% decreased at 2 h and 3 h post-LANH
infusion (Fig. 1).
|
). Three hours after
vessel dilator infusion, the circulating concentrations of CRH
were 58% below their baseline concentrations (Fig. 2). Kaliuretic
hormone, likewise, caused a significant (P < 0.001)
75% decrease in the circulating concentrations of CRH at the end of
its infusion (Fig. 2). Kaliuretic hormone’s decrease in the
circulating concentrations of CRH were slower than vessel dilator’s
with a 25% decrease vs. 74% decrease at 20 min for vessel
dilator (Fig. 2). The decrease in CRH secondary to kaliuretic hormone
also did not last as long as the decrease secondary to vessel dilator
(Fig. 2). The circulating concentrations of CRH had returned to
baseline by 3 h after kaliuretic hormone infusion (Fig. 2).
|
). ACTH was 70% (P <
0.001) below its baseline concentration at 60 and 90 min post-ANH
infusion before returning to its baseline at 3 h post-ANH infusion
(Fig. 3). Kaliuretic hormone also decreased the circulating
concentrations of ACTH, but its onset of action was slower without any
significant decrease in ACTH during the first 20 min of its infusion
(Fig. 3). ACTH, however, decreased 66% in the circulation at the end
of the kaliuretic hormone infusion and was 80% (P <
0.001) below its baseline concentration at 60 and 90 min postkaliuretic
hormone infusion (Fig. 3). ACTH then began to increase toward its
baseline concentration between 21/2 and 3 h after
kaliuretic hormone infusion (Fig. 3).
|
). LANH
decreased the circulating ACTH concentrations 81% during its infusion
(Fig. 4). ACTH was still 50% below baseline 3 h after LANH
infusion (Fig. 4). Vessel dilator, likewise, significantly
(P < 0.001) decreased ACTH (58%) circulating
concentrations during its infusion and ACTH was 58% below baseline at
2 and 21/2 h after vessel dilator infusion. Three hours after
cessation of the vessel dilator infusion, the circulating
concentrations of ACTH were still 53% below their preinfusion ACTH
levels (Fig. 4).
|
). The decrease in
plasma cortisol secondary to ANH (Fig. 5) lasted longer than the
decrease in ACTH (Fig. 3
) or CRH (Fig. 1). Three hours after ANH
infusion, plasma cortisol levels were 67% (P < 0.001)
below the preinfusion cortisol levels (Fig. 5). Kaliuretic hormone
decreased plasma cortisol levels 67% (P < 0.001)
during its infusion in healthy humans (Fig. 5). Kaliuretic hormone
effects on the modulation of plasma cortisol levels were long lasting,
with plasma cortisol levels being 72% (P < 0.001)
below the preinfusion values at 2 h postkaliuretic hormone
infusion and 67% below preinfusion levels at 3 h after cessation
of the kaliuretic hormone infusion (Fig. 5).
|
). Similar to its long-lasting effects
on ACTH (Fig. 4) and CRH (Fig. 2), vessel dilator decreased plasma
cortisol concentrations for 3 h after its infusion (Fig. 6). The
maximal decrease in plasma cortisol (i.e. 73%,
P < 0.001) secondary to vessel dilator was actually
3 h after stopping its infusion (Fig. 6). LANH decreased the
circulating concentrations of cortisol 68% (P <
0.001) during its infusion (Fig. 6). The maximal decrease in cortisol
(73%, P < 0.001) was at 2 and 2 1/2 h
post-LANH infusion (Fig. 6). LANH effects on the circulating
concentrations of cortisol were also long lasting, with plasma
cortisols being 64% (P < 0.001) and decreasing 3
h post-LANH infusion, compared with their preinfusion values (Fig. 6).
The infusion of vehicle (i.e. 0.9% saline) for 60 min
(n = 6) did not result in any decrease in CRH, ACTH, or
cortisol.
|
In the present investigation, LANH, vessel dilator, kaliuretic hormone, and ANH each decreased the circulating concentrations of CRH, ACTH, and cortisol in healthy humans. ANH has previously been shown to inhibit acetylcholine (1), and potassium (2) stimulated release of CRH from isolated hypothalami in vitro using similar concentrations of ANH as in the present investigation. One previous study in humans (3) suggested that ANH might be a CRH-inhibiting hormone (CRIF), but CRH was not actually measured in that study. ANH, somatostatin, dopamine, and pre–pro-TRH (178–199) have each been suggested as possible candidates for CRIFs, but as reviewed by Engler et al. (27), none of these substances have filled all of the criteria that Engler et al. have suggested that a CRIF should potentially possess. Of the above substances, Engler et al. (27) found that ANH had the most compelling evidence to date that a neuropeptide may inhibit ACTH release in man. But both Engler et al. (27) and Weidemann et al. (28) have stressed that ANH’s potential effects on CRH have been somewhat controversial because CRH has not been directly measured in previous ANH investigations. The present data demonstrating that ANH can markedly decrease the measured circulating concentration of CRH in humans are direct evidence for the suggestion that ANH is a CRIF in humans.
ANH and the three other peptide hormones that decreased the circulating concentration of CRH in the present investigation are synthesized in the hypothalamus with the hypothalamus being the region of the brain with the highest concentrations of ANH prohormone mRNA (17). All four of these peptide hormones are synthesized as part of the same ANH prohormone (29) and are neurotransmitters that are transported down cholinergic neurons (16). Long-acting natriuretic peptide, vessel dilator, and kaliuretic hormone as well as ANH fit one of the criteria of Engler et al. (27) for a CRIF in that they are present in nerve terminals in the media eminence (16). These three peptides are released simultaneously with ANH to various stimuli (16, 24, 25). These three peptide hormones have not as yet been investigated for their ability to inhibit release of basal ACTH secretion and POMC gene expression in vitro or bind to receptors in the anterior pituitary or investigated as to whether their respective antibodies increase basal ACTH secretion, which are the other criteria that Engler et al. (27) suggest that a CRIF should have. (ANH antibodies cause a four- to five-fold increase in ACTH (13, 14) and a three-fold increase in CRH (15) in the circulation of stressed rats.) The demonstration in the present investigation that four peptide hormones studied can directly decrease the circulating concentrations of CRF in humans, however, does suggest that these peptides may be CRIFs.
C-natriuretic peptide produced in a separate gene from the above peptide hormones (16) has exactly opposite effects to these peptide hormones in that it increases the circulating concentration of cortisol (28). Brain natriuretic peptide, synthesized in yet another gene (16), has no effect on circulating ACTH concentrations in humans (30), and in animals BNP reportedly increases CRH-like immunoreactivity rather than decreases CRH (31).
ANH decreased the circulating concentrations of ACTH, which remained
70% decreased for 90 min after cessation of the ANH infusion (Fig. 3).
Whether this decrease in the circulating concentrations of ACTH was
owing to the simultaneously observed decrease in CRH or a direct effect
on the pituitary release of ACTH could not be determined from the
present investigation. Previous investigations have demonstrated that
ANH can inhibit basal (4, 6) and CRH-stimulated release of
ACTH from cultured pituitary cells (5, 6). ANH has also
been shown in humans to decrease the CRH- and
vasopressin-stimulated release of ACTH and cortisol
(32). These studies suggest that ANH can inhibit CRH’s
effect on ACTH at the level of pituitary as well as decreasing CRH in
the hypothalamus (4, 5, 6).
ANH also reduced plasma cortisol concentrations in healthy humans in the present investigation. This result is similar to that found by Ohashi et al. (33), who found that plasma cortisol significantly decreased when ANH was given for 20 min at the same concentration as in the present investigation. LANH, vessel dilator, and kaliuretic hormone also decreased the circulating concentration of cortisol, which would further suggest that these four peptide hormones may be involved in an negative feedback regulation of cortisol. Dexamethasone and cortisol enhance the synthesis of the four atrial natriuretic peptides by upregulating their gene expression (34, 35). When these four peptide hormones increase in the circulation secondary to their release after their enhanced gene expression by cortisol (or with their infusion as in the present investigation), they in turn decrease the amount of cortisol in the circulation (i.e. completing the negative feedback loop). It would appear that this negative feedback loop may also involve CRH because CRH has been reported to enhance the secretion of ANH from isolated cardiomyocytes (36, 37).
In summary, the present investigation demonstrates that four peptide hormones (i.e. ANH, vessel dilator, LANH, and kaliuretic hormone) each decrease the circulating concentrations of CRH, ACTH, and cortisol. These data, in combination with previous in vitro data of others outlined above, further suggest that these four peptide hormones may be part of an intricate feedback system to participate in the regulation of cortisol concentrations via their ability to decrease the circulating concentration of CRH, which in turn results in a decrease in ACTH and cortisol.
Acknowledgments
We thank Charlene Pennington and Rose M. Overton for excellent secretarial and technical assistance, respectively. We thank Dr. George Rodriguez-Paz, Margret Douglass, R.N., and James R. Parks, R.N., for assistance with the cardiac peptide infusions.
Footnotes
This work was supported by in part by a Merit Review Grant from the United States Department of Veteran Affairs (to D.L.V.) and a grant-in-aid from the American Heart Association, Florida Affiliate (to D.L.V. and D.D.S.).
Abbreviations: a.a., Amino acid; ANH, atrial natriuretic hormone; BNP, brain natriuretic peptide; CRIF, CRH-inhibiting hormone; LANH, long-acting natriuretic hormone.
Received January 24, 2001.
Accepted May 14, 2001.
References
-atrial natriuretic polypeptide on
adrenocortical function in man. J Endocrinol 110:287–292This article has been cited by other articles:
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) and ANH (
) decrease the circulating
concentration of CRH. LANH and ANH were infused at 100 ng/kg BW·min
for 60 min, with the circulating concentration of CRH being
significantly (P < 0.001) decreased within 20 min
of beginning their respective infusions when evaluated by ANOVA. The
decrease in CRH secondary to LANH remained significant
(P < 0.01) for 3 h after stopping the LANH
infusion. The decrease in CRH secondary to ANH was significant
(P < 0.001) for 90 min after stopping its infusion
but by 2 h after ANH infusion, the CRH concentration became
nonsignificant, compared with its preinfusion values when evaluated by
ANOVA (n = 6 for each group).
)
decrease the circulating concentration of CRH when infused at 100
ng/kg·BW for 60 min. The decrease in CRH secondary to vessel dilator
was significant (P < 0.001) within 20 min of
starting the vessel dilator infusion, and the decrease in CRH
concentration secondary to vessel dilator remained significant
(P < 0.01) for 3 h after cessation of the
vessel dilator infusion when evaluated by ANOVA. The decrease in CRH
secondary to kaliuretic hormone was significant (P
< 0.001) throughout its infusion and for 2 h after infusion when
evaluated by ANOVA. The circulating concentration of CRH was not
significantly different from its preinfusion values 21/2 and
3 h after the kaliuretic hormone infusion when evaluated by ANOVA
(n = 6 for each group).
