Protein undernutrition during development and oxidative impairment in the central nervous system (CNS): potential factors in the occurrence of metabolic syndrome and CNS disease.

Mitochondria play a regulatory role in several essential cell processes including cell metabolism, calcium balance and cell viability. In recent years, it has been postulated that mitochondria participate in the pathogenesis of a number of chronic diseases, including central nervous system disorders. Thus, the concept of mitochondrial function now extends far beyond the common view of this organelle as the 'powerhouse' of the cell to a new appreciation of the mitochondrion as a transducer of early metabolic insult into chronic disease in later life. In this review, we have attempted to describe some of the associations between nutritional status and mitochondrial function (and dysfunction) during embryonic development with the occurrence of neural oxidative imbalance and neurogenic disease in adulthood.

The effect of glutamine supplementation and physical exercise on neutrophil function.

Glutamine is the most abundant free amino acid in the body. Its primary source is skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues. Several studies have shown that glutamine is important for rat and human neutrophil function and that these cells utilize glutamine at high rates. Physical exercise has also been shown to induce considerable changes in neutrophil metabolism and function. As neutrophils represent 50-60% of the total circulating leukocyte pool and play a key role in inflammation, both physical exercise and glutamine might be expected to regulate the inflammatory process. In this review, the changes in neutrophil function induced by physical exercise and glutamine supplementation are compared.

Molecular mechanisms of glutamine action.

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions.

Glutamine-dependent changes in gene expression and protein activity.

The functions of glutamine are many and include, substrate for protein synthesis, anabolic precursor for muscle growth, acid-base balance in the kidney, substrate for ureogenesis in the liver, substrate for hepatic and renal gluconeogenesis, an oxidative fuel for intestine and cells of the immune system, inter-organ nitrogen transport, precursor for neurotransmitter synthesis, precursor for nucleotide and nucleic acid synthesis and precursor for glutathione production. In the present review information on the mechanism of glutamine action is presented. This amino acid has been shown to regulate the expression of several genes (such as p47phox, p22phox, gp91phox, alpha-actin and fibronectin) and activate several proteins (such as ASK1, c-myc, c-jun and p70s6k).

cAMP inhibits natriuretic peptide receptor-B activity and increases C-type natriuretic peptide in FRTL-5 rat thyroid cells.

C-type natriuretic peptide (CNP) and its cognate guanylyl cyclase receptor, the natriuretic peptide receptor B (NPR-B) together constitute a regulatory system that controls cell function via the generation of intracellular cyclic GMP. In this report we have examined the role of cAMP signaling in the regulation of CNP and NPR-B activity in the FRTL-5 rat thyroid follicular cell line. As had been observed earlier with TSH, the cAMP mimetic, dibutyryl cAMP (dbcAMP; 1 mM) induced a significant reduction in CNP-stimulated cGMP generation that was first apparent after 6 h of treatment. The inhibitory effect of dbcAMP on NPR-B was dose dependent, with an EC50 of 0.2 mM. Pretreatment of FRTL-5 cells with either of two protein kinase A (PKA) inhibitors, KT-5720 and H-89, failed to curtail the dbcAMP reduction in NPR-B activity, suggesting that the cAMP pathway leading to inhibition of NPR-B is PKA independent. Whereas either a 30-min or a 24-h treatment with the protein kinase C-activator phorbol myristate acetate failed to alter maximal levels of CNP-stimulated cGMP, a 24-h exposure to the calcium ionophore A23187 reduced CNP-stimulated cGMP to about one-third of control. Pretreatment of FRTL-5 cells with the cell-permeable calcium chelator 1,2 bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, tetraacetoxymethyl ester completely abrogated the cAMP-induced reduction of CNP-stimulated cGMP. Real-time PCR showed no effect of dbcAMP on NPR-B transcript at 3 and 6 h, but indicated a 40% reduction in transcript by dbcAMP at 24 h. In contrast, real-time PCR indicated a 5-fold increase in CNP transcript at 3 h, reaching 15.4-fold above control at 6 h in cells treated with dbcAMP. In addition, immunofluorescence staining of FRTL-5 cells with a specific antibody for CNP-22 showed the presence of cytoplasmic CNP that was up-regulated by incubation with either TSH or dbcAMP. These results suggested that cAMP signaling regulates the natriuretic peptide system in rat thyroid cells by increasing CNP expression, and reducing NPR-B activity. This latter action of cAMP appears to be both PKA independent and calcium dependent, and provides support for a dominant role for calcium in the regulation of NPR-B in the rat thyroid.

Atrial natriuretic factor and C-type natriuretic peptide induce retraction of human thyrocytes in monolayer culture via guanylyl cyclase receptors.

The natriuretic peptides signal through three receptor subtypes, of which two (NPR-A and NPR-B) are membrane-bound guanylyl cyclases for which the principal ligands are respectively atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP). In the human thyroid cell, a third receptor, NPR-C, has been implicated in the regulation of thyroglobulin, but functional roles for NPR-A and NPR-B have not yet been defined. In the present study we used RT-PCR to identify transcripts of all three receptor subtypes, both in human thyroid and in HTU-5 cells, a long-term culture of thyroid-derived cells. Both ANF and CNP induced a twofold increase in intracellular cGMP content in HTU-5 cells. Morphologic changes (a significant increase in cells of the retracted phenotype) were observed in ANF- and CNP-treated cells within 3 and 5 h of treatment respectively. Significant increases in retracted cell number were induced by ANF and CNP, but not the NPR-C-specific ring-deleted ANF analog, C-ANF(4-23), during a 15-day treatment. All three natriuretic peptides, however, induced a small (15-20%) but significant (P<0 small middle dot001) increase in DNA content per well. The stable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), also increased the number of retracted HTU-5 cells, and was equipotent with the cAMP analog, 8-BrcAMP, in this effect. The cGMP-dependent protein kinase inhibitor, KT5823, however, had no significant effect on the ANF-induced increase in numbers of retracted cells. These results suggest that the actions of NPR-A and NPR-B, functional receptors in the human thyroid cell, may in part be mediated by cGMP-induced alterations in the cytoskeleton.

Thyroglobulin increases cell proliferation and suppresses Pax-8 in mesangial cells.

Thyroglobulin (Tg), has recently been identified as a transcriptional regulator of thyroid-restricted genes. The extrathyroidal expression of several of these genes (including the transcription factor Pax-8) together with the occurrence of specific Tg binding sites suggests a secondary role for Tg as a circulating hormone. In this study, we demonstrate using Northern analysis that Pax-8 is expressed in the mouse mesangial cell, and that its transcript levels are suppressed by Tg. These cells also express an asialoglycoprotein receptor, a receptor involved in Tg endocytosis in the thyroid, and a Tg transcript smaller than the 8.3-kb thyroidal form. Reverse transcriptase PCR showed that suppression of Pax-8 by Tg is correlated with reduced expression of bcl-2 apoptosis suppressor. Tg, but not triiodothyronine (T(3)) significantly increased MC proliferation above control as determined by DNA content of MC cultures. The effect of Tg on proliferation was not duplicated by either bovine serum albumin, gamma-globulins, lactoferrin, or the ASGPR-specific ligand,orosomucoid. These results suggest a possible endocrine role for Tg in regulating both Pax-8 related gene transcription and cell division in the mesangial cell.

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C).

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [125I] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200-300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype.

Renal expression of two 'thyroid-specific' genes: thyrotropin receptor and thyroglobulin.

Numerous renal abnormalities accompany thyroid disease, most of which have been ascribed to the effects of thyroid hormone on renal metabolism. In the present report, we investigate the renal expression of the nominally thyroid-specific proteins, thyroid-stimulating hormone (TSH) receptor (TSHR) and thyroglobulin (Tg), as potential links between renal and thyroid function. The expression of TSHR has been identified in several extrathyroidal tissues, but its presence in the kidney remains controversial. We have used reverse-transcriptase polymerase chain reaction and DNA sequencing to demonstrate the presence of TSHR transcript in human and mouse kidney, in a primary culture of human kidney, and in a green monkey kidney epithelioid cell line. Furthermore, human kidney cells responded to TSH with a 2.5- fold increase in intracellular cyclic adenosine monophosphate, suggesting the presence of functional TSHR protein. Comparison of renal expression of TSHR in a bovine growth hormone transgenic mouse model of progressive glomerulosclerosis with control mice suggested increased TSHR transcript in the renal cortex of transgenic animals. TSHR transcript was also detected in mouse mesangial cells in vitro which responded to TSH with significant increases in the formation of three-dimensional hillhocks. Polymerase chain reaction also confirmed the presence of Tg transcript in human and mouse kidneys and in mouse mesangial cells, but no effect of either TSH or cyclic adenosine monophosphate on Tg transcript levels could be discerned. Immunofluorescent staining with a monoclonal anti-Tg antibody identified positive staining in the cytoplasm of mesangial cells. These data suggest that the kidney is capable of expressing the thyroid-specific genes, TSHR and Tg, which could conceivably mediate effects of thyroid disease in the kidney.

Thyrotropin regulation of cyclic adenosine monophosphate production in human coronary artery smooth muscle cells.

Thyroid disease has been associated with the occurrence of pathophysiologic changes in the vasculature that may result in part from altered serum thyroid hormone and serum lipid levels. Thyrotropin (TSH) levels are also altered in thyroid disease, but a direct effect of TSH on vascular smooth muscle has not previously been considered. In the present study, human coronary artery smooth muscle cells (CASMC) were induced into two morphologically distinct forms by culturing in either (1) growth factor supplemented, 0.5% serum medium (SmGM-3) or (2) basal medium (SmBM) plus 10% fetal bovine serum (FBS). Intracellular cyclic adenosine monophosphate (cAMP) accumulation was determined by radioimmunoassay after exposure to increasing doses of bovine TSH. Cells grown in SmBM/10% FBS for 3 days exhibited a dose-dependent increase in intracellular cAMP that reached a level 10 times higher than baseline at the highest dose examined (100 mIU/mL). In contrast, cells grown in SmGM-3 medium exhibited no change in intracellular cAMP on exposure to increasing TSII. Low serum (0.5% FBS) reduced the ability of TSH to stimulate cAMP above the control value in CASMC. Pretreatment of CASMC with either transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha) lowered basal levels of cAMP production, but did not inhibit the ability of TSH to stimulate cAMP production. Human, but not rat aortic smooth muscle cells in culture also responded to TSH with a significant increase in cAMP. The results of this study suggest that TSH may exert direct effects on vascular smooth muscle mediated by adenylate cyclase activation that could conceivably affect the progression of vascular disease associated with thyroid dysfunction.

Regulation of natriuretic peptide receptors by thyrotropin in FRTL-5 rat thyroid cells: evidence for nonguanylate cyclase atrial natriuretic factor-binding sites in cells lacking the natriuretic peptide receptor C.

Natriuretic peptide receptors (NPR) are expressed in thyroid-derived cells, including the rat FRTL-5 thyroid cell line. We have previously demonstrated that atrial natriuretic factor (ANF) binding consistent with the NPR-A receptor is significantly increased in FRTL-5 cells cultured in the presence of TSH. The purpose of the present study was to determine whether TSH treatment, therefore, results in higher levels of ANF-induced intracellular cGMP, and whether TSH elicits similar effects on cGMP signaling through the NPR-B receptor. We now show that contrary to expectation, long term exposure to 1 mIU/ml bovine TSH (6H medium) does not significantly alter maximal ANF-induced cGMP formation. Moreover, TSH treatment decreased C-type natriuretic peptide (CNP)-induced cGMP generation in FRTL-5 cells, suggesting a down-regulation of NPR-B. A similar effect of TSH on ANF- and CNP-induced cGMP was observed in FRTL cells, the precursor of the FRTL-5 cell line. Scatchard analysis of [125I]ANF binding in TSH-treated (6H) FRTL-5 cultures indicated a 5.6-fold increase in high affinity ANF-binding sites compared with TSH-deficient (5H) cultures [binding capacity (Bmax) of 6H cells, 227.2 +/- 33.7 fmol/mg protein; Bmax of 5H cells, 40.2 +/- 4.7 fmol/mg protein]. The effect of TSH on [125I]ANF binding was mimicked by forskolin and (Bu)2cAMP, indicating receptor up-regulation via a cAMP pathway. High affinity [125I]CNP-binding sites were present in much lower abundance (Bmax of 5H, 0.80 +/- 0.06 fmol/mg protein), and no effect of TSH treatment on them could be demonstrated. However, low affinity [125I]CNP binding was increased by TSH. RT-PCR confirmed the presence of both NPR-A and NPR-B transcripts in FRTL-5 cells and showed that TSH treatment significantly decreased NPR-B, but not NPR-A. NPR-C transcript was not detectable by RT-PCR in FRTL-5 cells cultured in high TSH medium, suggesting that the ANF-binding sites increased by TSH are not NPR-C. Both CNP and ANF transcript were also expressed in FRTL-5 cells, and CNP was increased by TSH. Together the data support the down-regulation of functional NPR-B and no change in functional NPR-A by TSH. The vast majority of ANF-binding sites in FRTL-5 cells, therefore, are not coupled to cGMP production and may represent a novel or altered form of NPR that is regulated by TSH independently of NPR-A and NPR-B.

Differential expression of thyrotropin receptor mRNA in the porcine heart.

Thyrotropin receptor (TSHR) mRNA expression has previously been detected in human heart, suggesting a possible role for the receptor in cardiac function and pathophysiology. In the present study we examined the regional distribution of TSHR mRNA in pig heart to map potential cardiac sites of TSH action. Polyadenylated mRNA extracted from thyroid, atria, ventricles, aorta, coronary arteries, epicardial fat, and purified preparations of atrial and ventricular cardiomyocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) using primers designed to amplify a 311 base pair (bp) DNA segment of the human TSHR. After reverse transcription of 100 ng mRNA, cDNA was amplified by PCR using TSHR primers and compared by electrophoresis on 2% agarose gels. Relative levels of TSHR cDNA (normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) were as follows: Coronary arteries, epicardial fat > right atrium > left atrium > right ventricle, aorta > left ventricle, ventricular cardiocytes. In contrast to ventricular cardiocytes, purified atrial cardiocytes expressed levels of TSHR mRNA readily detectable with RT-PCR. These findings demonstrate that TSHR mRNA expression in porcine heart varies regionally, and furthermore suggest that areas of highest expression (coronary arteries, adipose tissue, right atrium) are potential sites for a functional or pathologic role of the TSHR.

Cardiomyopathy associated with Graves' disease.

Cardiovascular changes associated with Graves' disease are generally considered to be secondary to the increased levels of thyroid hormone. We describe a case of Graves' disease in a 25-year-old man, who developed cardiomyopathy with severe heart failure. Pathological examination of the myocardial biopsies showed fibroblast infiltration and degenerative changes. After the cardiomyopathy subsided the patient developed a goitre and signs of hyperthyroidism, followed by Graves' ophthalmopathy, which was treated successfully with a combination of high-dose corticosteroids and orbital radiotherapy. These findings suggested a common pathogenesis for the cardiomyopathy and ophthalmopathy, and prompted us to investigate the expression of TSH receptor (TSH-R) in human heart. TSH-R mRNA was identified in human heart using the reverse transcriptasepolymerase chain reaction (RT-PCR) and DNA sequencing. Taken together, these data suggest that autoimmunity against the TSH-R might contribute to both the cardiomyopathy and ophthalmopathy in similar cases of Graves' disease.

C-type natriuretic peptide (CNP) increases [125I]ANF binding to FRTL-5 rat thyroid cells by increasing ANF receptor affinity.

To examine a possible role for C-type natriuretic peptide (CNP) in the thyroid, we studied the ability of this peptide to compete with atrial natriuretic factor (ANF) binding to FRTL-5 rat thyroid cells. Rather than competing for ANF binding, CNP significantly elevated [125I]ANF binding above control at both 23 degrees and 2 degrees C. The increase in ANF binding was due largely to a threefold increase in receptor affinity in the presence of CNP (control, Kd = 8.7 nM; 1 microM CNP, Kd = 3.1 nM). Despite the failure to compete for ANF binding, CNP was almost as effective as ANF at inducing cGMP production in FRTL-5 cells. Competition binding studies using [125I]CNP indicated the presence of a relatively low-affinity site for CNP (Kd = 77 nM) that bound ANF with equal affinity. These results show for the first time that ANF receptor binding can be positively regulated by the related natriuretic peptide, CNP.

Atrial natriuretic factor (ANF) binds to thyrotropin-regulated receptors in FRTL-5 cells and increases thyroglobulin mRNA.

Thyrotropin (TSH) regulation of atrial natriuretic factor (ANF) receptors was studied in the rat thyroid follicular cell line, FRTL-5. Exposure of FRTL-5 cells to 1 mU/ml TSH for 7 days resulted in a tenfold increase in ANF receptors (Bmax = 188 fmol/mg protein) compared with control (Bmax = 18 fmol/mg protein), without affecting binding affinity. An identical treatment of porcine thyrocytes with TSH resulted in a 50% decrease in ANF binding sites. Displacement binding studies indicated that > 80% of the ANF receptors in FRTL-5 cells belong to the ANF-R1 (guanylate cyclase-coupled) receptor subtype. By contrast, > 98% of the ANF receptors in porcine thyrocytes were of the ANF-R2, or clearance, receptor subtype. Intracellular cGMP content was increased thirty-sixfold in FRTL-5 cells by 1 microM ANF, but only 2.5-fold in porcine thyrocytes. cAMP levels were unaffected by ANF in either cell type. Northern blot analysis of poly A mRNA extracted from FRTL-5 cells incubated 2 days in the presence of 100 nM ANF indicated a twofold increase in thyroglobulin mRNA content compared with control. These findings suggest that the ANF-R1 receptor, preferentially expressed in FRTL-5 cells and regulated by TSH, might play a role in regulating thyroid hormone production.

Secretion of immunoreactive ANF from FRTL-5 rat thyroid cells: regulation by calcium ionophore A23187.

Atrial natriuretic factor-like immunoreactivity (ir-ANF) was characterized in a continuous line of rat thyroid follicular cells (FRTL-5) and the influence of the calcium ionophore A23187 on ir-ANF secretion was examined. Ir-ANF was identified by immunohistochemical staining as primarily reticular and juxtanuclear in short-term cultures, and more peripheral and granular in longer-term cultures, suggesting a process of ir-ANF packaging into secretory granules. The accumulation of ir-ANF granules was dependent upon the presence of thyrotropin (TSH) in the medium. Secreted ir-ANF was characterized using reversed-phase, high-performance liquid chromatography (RP-HPLC) and radioimmunoassay as a single peak eluting one fraction earlier than 125I-labeled rat ANF (99-126) (i.e., circulating atrial ANF) included as an internal standard. A23187 treatment of cells exhibiting primarily reticular ir-ANF caused a change to a pattern of more distinct, peripherally localized granules. This change occurred within 1 h after A23187 treatment and was dependent on the presence of Ca2+ in the medium. In cultures containing primarily ir-ANF granules, A23187 (0.5 micrograms/ml) induced a peripheral translocation of the granules at 30 min and a complete degranulation by 7 h. Enzyme-linked immunoadsorbent assay (EIA) confirmed a dose-dependent effect of A23187 on ir-ANF release into the medium. These results suggest that some of the effects of Ca2+ in the thyroid could be ascribed to its mobilization and release of ir-ANF, which in turn may have autocrine effects on thyroid follicular cells.

Thyroidal ANF: a possible mediator of autocrine regulation in the porcine thyroid gland.

Atrial natriuretic factor immunoreactivity (ir-ANF) was examined in thyroid tissue sections and cultured thyroid cells using immunohistochemical staining with a monoclonal anti-ANF antibody. Localization of ir-ANF in perinuclear granules in cultured cells and in the basal region of follicular cells in sectioned tissue suggests that ir-ANF is a basally secreted product. Thyroidal ir-ANF was also characterized using reversed phase high performance liquid chromatography (RP-HPLC) of acidic thyroid extracts. An ir-ANF peak coeluting with synthetic rat ANF(99-126) suggests that thyroidal ir-ANF may be identical in form to circulating atrial ANF. However, the detection of ir-ANF in cultured thyroid cells confirms that the immunoreactivity is locally produced. Saturation analysis revealed high affinity ANF receptors (Kd = 0.1 nM; MBC = 17.2 fmol/mg protein) on these cultured cells, and a competition study demonstrated the ability of extracted thyroidal ir-ANF to inhibit 125I rat ANF binding to the membrane receptors. The evidence presented here suggests that ir-ANF in the thyroid may be secreted locally to exert an autocrine effect on neighboring follicular cells.

Amiloride differentially modulates ANP binding in human thyroid cells and bovine endothelial cells.

The diuretic and sodium channel inhibitor, amiloride, has been shown to increase atrial natriuretic peptide (ANP) binding several fold in certain cell types, but in other tissues it causes only marginal increases in specific ANP binding. In the present report we compare the effects of amiloride on ANP binding in bovine endothelial cells and human thyroid-derived cells, two cell types which differ in their predominant ANP receptor subtype. We found that amiloride (10(-3) M) increased specific [125I]ANP binding to 750% above control in endothelial cells, but among several thyroid cultures tested the maximal increase in ANP binding with amiloride was only 23% above control. Moreover, most of the thyroid cultures showed decreased ANP binding in the presence of amiloride. The increased ANP binding in endothelial cells exposed to amiloride is best explained by an increased affinity of the receptor for its ligand since the drug lowered the Kd of ANP binding from 0.73 nM to 0.16 nM without affecting the receptor binding capacity. The degree of amiloride enhancement of ANP binding in endothelial cells is increased with time in culture (200% above control at 5 days, 750% above at 30 days) suggesting the increase of an amiloride-sensitive receptor relative to an amiloride-insensitive receptor. The fact that the amiloride-induced decrease in ANP binding in thyroid cells was not exacerbated by pre-incubation with amiloride suggested that the observed amiloride effect was not due to increased receptor internalization with the drug. These results support a hypothesis that ANP receptor subtypes associated with separate signal transduction mechanisms might be modulated in an opposite manner by the binding of amiloride.

Immunoreactive atrial natriuretic peptide in the thyroid gland.

Human thyroid follicles and primary cell cultures derived from them demonstrated atrial natriuretic peptide (ANP)-like immunoreactivity when stained with a monoclonal antibody raised against rat alpha-ANP (ANP 1-28). In thyroid sections the staining was most intense in the tall cuboidal epithelium of small follicles. The intracellular distribution of immunoreactive (ir)-ANP in primary cultures of thyroid follicular cells consisted of discrete granules with a largely perinuclear distribution. The granule density increased with time in culture but was unaffected by exogenous ANP, suggesting an intrinsic synthesis of the immunoreactivity. Thyroid stimulating hormone (TSH; thyrotropin) failed to alter the distribution of ir-ANP after either short-term (6 h) or long-term (1-12 day) exposure. Epinephrine or norepinephrine treatment, however, caused a reduction in the ir-ANP granularity compared with controls in what might represent a stimulated release of the immunoreactivity. The present results suggest that the peptide ANP coexists with thyroid hormones in follicular cells and that the two endocrine activities might be under separate control mechanisms.