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University of South Florida Cardiac Hormone Center; Departments of Biochemistry, Molecular Biology, 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., Department of Endocrinology, Diabetes, and Metabolism, James A. Haley Veterans Hospital 151, 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 (ANPs) consisting of amino acids 1–30 [i.e. long-acting natriuretic hormone (LANH)], 31–67 (vessel dilator), 79–98 (kaliuretic hormone), and 99–126 (atrial natriuretic hormone [ANH]) of the 126-amino acid ANH prohormone decrease the circulating concentrations of total and free T4 and/or free T3 in healthy humans (n = 30). Vessel dilator, kaliuretic hormone, LANH, and ANH decreased the circulating concentrations of total T4 by 61%, 58%, 47%, and 55% and of free T4 by 60%, 67%, 79%, and 79%, whereas free T3 decreased 72%, 67%, 71%, and 67% (P < 0.05 for each), respectively, when infused at 100 ng/kg BW·min for 60 min. Vessel dilator, kaliuretic hormone, LANH, and ANH simultaneously increased circulating TSH concentrations 4- to 12.5-fold (P < 0.004). The decreases in T4 and T3 with reciprocal increases in TSH lasted 2–3 h after cessation of the respective ANP infusions. The reciprocal increase in TSH with the decreases in T4 and T3 suggests that their modulation of T4 and T3 concentrations occurs in the thyroid rather than in the pituitary or hypothalamus, because TSH would be decreased in the circulation if their inhibitory effects were in either the hypothalamus or pituitary.
T4 AND T3 increase atrial natriuretic hormone (ANH) prohormone mRNA in primary cardiac myocyte cultures, indicating that both T4 and T3 enhance ANH prohormone gene expression (1, 2, 3, 4). The products of this prohormone’s gene expression are four peptide hormones consisting of amino acids (aa) 1–30 [pro-ANH-(1–30); long-acting natriuretic hormone (LANH)], aa 31–67 [pro-ANH-(31–67); vessel dilator], aa 79–98 [pro-ANH-(79–98); kaliuretic hormone), and aa 99–126 [pro-ANF-(99–126); ANH) of the 126-aa prohormone (5). With respect to clinical correlation, hypothyroid subjects have decreased circulating concentrations of LANH, vessel dilator, and ANH (collectively termed ANPs) (6, 7, 8, 9, 10). On the other hand, hyperthyroid individuals have 2- to 4-fold increased circulating concentrations of ANPs compared with healthy subjects (6, 7, 8, 9, 10). These peptide hormones also increase proportionally in the circulation of hypothyroid subjects with increasing replacement doses of L-T4 of 50 and 100 µg/d (10). When hypothyroid patients become euthyroid with T4 treatment, the circulating levels of ANPs increase to those of normal healthy adults (10).
ANH, on the other hand, inhibits the TSH-induced increase of radioiodine levels in the mouse (11). Binding sites for ANH have been found on human thyroid follicular cells in culture (12, 13). These studies suggest that ANH and possibly the other ANPs may inhibit thyroid hormone secretion from the thyroid gland. The present investigation was designed to determine whether infusion of ANH, vessel dilator, LANH, and/or kaliuretic hormone decrease the circulating concentrations of total and free T4 and/or free T3. When each of these peptide hormones decreased the circulating concentration of free and total T4 and free T3, the concentration of TSH was measured in the same plasma samples to help determine whether the decreases in T4 and T3 in the circulation were due to a direct effect(s) of these ANPs on the thyroid and/or possibly by decreasing the circulating concentration of TSH.
Subjects and Methods
Experimental subjects
Thirty healthy subjects (15 men and 15 women; aged 20–58 yr;
average, 32 yr; all normotensive with blood pressures <125/80 mm Hg)
were studied. These subjects had heart rates ranging from 56–80
beats/min, with respiration rates between 12–14/min. These 30
volunteers were divided into 5 similar groups with 6 individuals in
each group. The age, weight, blood pressure, and heart rates for each
group are outlined in Table 1
. None of
the volunteers had any known disease. 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. 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
(14).
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After obtaining written informed consent, an Insyte-w 20-gauge 1.5-in catheter (Becton Dickinson Infusion Therapy Systems, Inc., Sandy, UT) 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 samples were obtained every 20 min during the infusion and at 30-min intervals during the 1-h baseline and 3-h 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 ANPs, because the rate of release of the N-terminal ANH prohormone peptides from the atrium of dog heart with physiological stimuli is 138–292 ng/kg BW·min, whereas the release rate of ANH is 76 ng/kg BW·min (15). 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/liter·kg BW for ANH, LANH, vessel dilator, and kaliuretic hormone, respectively.
Measurement of total T4
Each of the blood samples to measure T4 and TSH were collected into chilled 5-ml ETDA tubes to prevent the proteolytic breakdown of TSH. T4 was measured using a solid phase 125I RIA from Diagnostic Products (Los Angeles, CA) with all measurements performed in the same assay. This assay is highly specific for T4 with the following cross-reactivities: L-T4, 100%; D-T4, 64%; tetraiodothyroacetic acid, 104%; triiodo-L-thyronine, 2%; triidodothyroacetic acid, 2%; monoiodotyrosine, undetectable; diiodo-L-tyrosine, undetectable; methimazole, undetectable; 5,5'-diphenylhydantoin, undetectable; phenylbutazone, undetectable; and 6-n-propyl-2-thiouracil, undetectable. The intra- and interassay coefficient(s) of variation for the T4 assay were 3.2% and 8.2%, respectively.
Measurement of free T4
Free T4 was measured with a solid phase 125I RIA from Diagnostic Products. The euthyroid range of free T4 is 10.3–25.7 pmol/liter, and this assay’s detection limit is 1.3 pmol/liter. The intra- and interassay coefficients of variation for free T4 assay were 5% and 8%, respectively. Importantly, the analog tracer in this free T4 assay does not bind to T4-binding globulin (TBG).
Measurement of free T3
Free T3 was measured with a solid phase 125I RIA from Diagnostic Products. The euthyroid range of free T3 is 2.2–6.8 pmol/liter. This assay cross-reacts only 0.00008% with T4. The T3 analog tracer does not bind to either TBG or albumin. The intra- and interassay coefficients of variation for the free T3 assay were 5% and 6.9%, respectively.
Measurement of TSH
TSH was measured using an immunoradiometric assay from Diagnostic Products in the same plasma samples collected from 0–300 min (n = 30). This TSH assay can detect as little as 0.03 µIU/ml TSH and has less than 0.1% cross-reactivity with other glycoprotein hormones, such as FSH, LH, and hCG. There is excellent parallelism (92–113%) of standards and unknowns in this assay. The intra- and interassay coefficients of variation for the TSH assay were 3.4% and 5.4%, respectively.
Statistical analysis
The data obtained in this investigation are presented as the mean ± SE. Differences in T4, T3, and TSH measurements between subjects or groups of subjects were evaluated by one-way ANOVA. Measurements of T4, T3, and TSH obtained in the same subjects over time were evaluated by two-tailed t test of the difference between the means of paired samples. To be considered statistically significant, we required P < 0.05 (95% confidence).
Results
ANH decreased the circulating total T4
concentration 39% during the first 20 min of its infusion with
T4, being 42% (P < 0.07) below
its baseline concentration at the end of its infusion (Fig. 1). The maximal decrease (55%;
P < 0.03)) in the circulating total
T4 concentration secondary to ANH occurred 2
h after ANH infusion was stopped (Fig. 1). Kaliuretic hormone also
decreased the circulating total T4 concentration,
but its onset of action was slower, without any significant decrease in
T4 during the first 20 min of its infusion (Fig. 1). Total T4, however, decreased 51% in the
circulation at the end of the kaliuretic hormone infusion and was 58%
(P < 0.03) below its baseline concentration at 30 min
postinfusion (Fig. 1). Total T4 then began to
increase toward its baseline concentration, but was still 33% below
its baseline concentration 3 h after ceasing the kaliuretic
hormone infusion (Fig. 1). Vessel dilator decreased the circulating
total T4 concentrations 61% (P
< 0.05) during its infusion (Fig. 1). LANH, likewise, had its maximal
effect at the end of its infusion, decreasing the total
T4 circulating concentration 47%
(P < 0.04; Fig. 1). The infusion of vehicle
(i.e. 0.9% saline) for 60 min (n = 6) did not result
in any decrease or increase in T4,
T3, or TSH.
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) followed a pattern similar to the
decrease in total T4 (Fig. 1
). The maximal
decreases in free T4 secondary to vessel dilator,
kaliuretic hormone, LANH, and ANH were 60%, 67%, 79%, and 79%,
respectively. As shown in Fig. 2, their effects on free
T4 began within 20 min and were relatively
long-lasting, with a significant decrease (P < 0.05)
in free T4 for 2–2.5 h after stopping the
respective peptide hormone infusions. There was no change in the
urinary concentration of free T4 secondary to
these peptide hormones when measured at the same time points.
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). The maximal decreases in free
T3 secondary to vessel dilator, kaliuretic
hormone, LANH, and ANH were 72%, 67%, 71%, and 67%, respectively.
There was no change in the urinary concentration of free
T3 secondary to these peptide hormones.
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). The increase in plasma
TSH secondary to ANH was maximal at the end of the ANH infusion and
began to decrease in the circulation with stopping the ANH infusion
(Fig. 4). Three hours post-ANH infusion, plasma TSH levels were 394%
(P = 0.007) above the preinfusion TSH levels (Fig. 4).
Kaliuretic hormone increased plasma TSH levels 542% (P
< 0.002) during its infusion in healthy humans (Fig. 4). The effect of
kaliuretic hormone on the modulation of plasma TSH levels was
long-lasting, with plasma TSH levels being 10- to 12.5-fold increased
(P < 0.001) above the preinfusion values during the
first 90 min post-kaliuretic hormone infusion and 6.7-fold increased
above preinfusion levels 2 h after cessation of the kaliuretic
hormone infusion (Fig. 4).
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).
Similar to its long-lasting effects on T4 and
T3 (
Figs. 1–3), vessel dilator-induced increased
plasma TSH concentrations were still 5.92- and 2.85-fold increased at
2.5 and 3 h, respectively, after its infusion (Fig. 4). LANH
increased the circulating concentration of TSH 368% (P
< 0.004) during its infusion (Fig. 4). The maximal increase in TSH
(559%) occurred 2.5 h post-LANH infusion (Fig. 4),
i.e. LANH’s effects on the circulating concentrations of
TSH were also long-lasting and correlated with LANH’s ability to
decrease circulating T4 and
T3 levels (
Figs. 1–3). Discussion
ANH, LANH, vessel dilator, and kaliuretic hormone, which each circulate in humans (16, 17), decreased the circulating concentrations of total and free T4 and free T3 in human volunteers in the present investigation. The respective decreases in T4 and T3 produced by these peptide hormones were sufficient to cause a reciprocal increase in TSH. Thus, the decreases in T4 and T3 in these human subjects caused the pituitary to release more TSH to try to overcome these decreases. The findings of the present investigation indicate that there are at least four other endogenous peptide hormones in addition to TSH that can modulate the circulating concentrations of T4 and T3.
ANH-, LANH-, vessel dilator-, and kaliuretic hormone-induced decreases in the circulating concentration of T4 appear to be secondary to a direct effect(s) on the thyroid gland and not via an effect(s) on the hypothalamus or pituitary of the hypothalamic-pituitary-thyroid axis. If these peptide hormones were decreasing the release of TRH in the hypothalamus or of TSH from the pituitary, then the circulating concentration of TSH should have been decreased in either case. As TSH was not decreased but, rather, was increased (in an apparent response to the decreases in T4 and T3), this observation would strongly suggest that the ability of these four peptide hormones to decrease T4 in the circulation is via their ability to inhibit the release of thyroid hormones from the thyroid gland. ANH has been shown to inhibit thyroid hormone secretion in the mouse (11). Receptors for ANH have been found on human thyroid follicular cells (12, 13). The rapidity with which T4 and T3 decreased in the circulation suggests that these peptide hormones inhibit Tg proteolysis of stored T4 and T3, which are released together (18), rather than interfering with their synthesis, which would usually necessitate a longer period to occur.
The ability of these peptide hormones to decrease free T3 and T4 suggests that the observed decrease(s) in total T4 secondary to these peptide hormones was not due to an effect on TBG, as the total T4 concentrations paralleled the free T4 concentrations, which do not bind to TBG. Further, if the decrease in total T4 had been due to an effect on TBG by these peptide hormones, the TSH would also have remained in the normal range and would not have become increased, which occurs when free T3 and T4 are decreased as found in the present investigation. The free T3 and free T4 decreases paralleling each other further suggests that the effects of these peptide hormones to decrease total T4 were not due to enhanced peripheral conversion of T4 to T3, where T3 would have increased in the circulation. There was no increased excretion of free T3 or T4 into the urine, indicating that the decrease in these thyroid hormones in the circulation was not due to enhanced degradation secondary to these peptide hormones. Using free T4, free T3, total T4, and TSH measurements in combination with the above previous in vitro studies suggests that the effects of these peptide hormones are direct on the thyroid. With respect to their possible direct effects on the thyroid, it is of interest that immunohistochemistry studies have revealed that ANH staining within the thyroid is most intense in the tall cuboidal epithelium of small follicles (19). The distribution of ANH within the follicular cells parallels that of Tg, except that Tg is distributed throughout the cytoplasm, whereas ANH is confined to distinct granules within the follicular cells (19).
The data of the present investigation suggest that LANH, ANH, vessel dilator, and kaliuretic peptide may be important in the negative feedback regulation of T4 and T3. The synthesis of these four ANPs is enhanced by T4 and T3 by up-regulating their gene expression (1, 2, 3, 4). LANH, vessel dilator, and kaliuretic peptide are derived from the same prohormone as ANH and synthesized by the same gene (20). T4 increases the circulating concentrations of these ANPs in subjects with hypothyroidism (10), indicating clinical relevance of the up-regulation of the ANH gene (1, 2, 3, 4). This gene is modulated by hypothyroidism and hyperthyroidism (4). When LANH, vessel dilator, kaliuretic peptide, and ANH are increased in the circulation after their enhanced gene expression by T3 and T4 (or with their infusion as in the present investigation), they, in turn, decrease the amounts of T4 and T3 in the circulation, completing the negative feedback loop.
In summary, the present investigation demonstrates that four peptide hormones, i.e. ANH, LANH, vessel dilator, and kaliuretic hormone, each decrease the circulating concentrations of T4 and T3. The reciprocal increase in TSH and the decreases in T4 and T3 by these four peptide hormones suggests that their modulation of T4 and T3 concentrations occurs in the thyroid, rather than in the pituitary or hypothalamus, as TSH would be decreased in the circulation if their inhibitory effects were in either the hypothalamus or pituitary. The present data in combination with previous data indicating that thyroid hormones increase the synthesis of the ANPs further suggest that these four peptide hormones function as negative feedback regulators of T4 and T3.
Acknowledgments
We thank Charlene Pennington and Rose M. Overton for excellent secretarial and technical assistance, respectively. We thank Dr. George Rodriguez-Paz, Margaret Douglass, R.N., and James R. Parks, R.N., for their assistance with the cardiac peptide infusions.
Footnotes
This work was supported in part by Merit Review Grants from the U.S. Department of Veteran Affairs (to D.L.V. and W.R.G.) and grant-in-aids from the American Heart Association, Florida Affiliate (to D.L.V., W.R.G., and D.D.S.).
Abbreviations: aa, Amino acids; ANP, atrial natriuretic peptide; LANH, long-acting natriuretic hormone; TBG, T4-binding globulin.
Received April 18, 2001.
Accepted August 14, 2001.
References
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), vessel dilator (
), and
kaliuretic hormone (
) decrease the circulating concentration of
total T4. ANH, LANH, vessel dilator, and kaliuretic hormone
were infused at 100 ng/kg BW·min for 60 min, with the circulating
concentrations of total T4 being significantly
(P < 0.05) decreased within 20 min of beginning
the ANH, LANH, and vessel dilator infusions, but not significantly
decreased until 40 min with the infusion of kaliuretic hormone when
evaluated by ANOVA. The decreases in T4 secondary to those
in kaliuretic hormone and ANH were significant (P
< 0.05), whereas the decreases secondary to LANH and vessel dilator
were not significant 3 h after stopping the infusions compared
with their preinfusion values when evaluated by ANOVA. n = 6 for
each group.
