Insulin and epidermal growth factor. Human fibroblast receptors related to deoxyribonucleic acid synthesis and amino acid uptake.

Receptors for insulin and epidermal growth factor (EGF) have been studied in confluent cultured intact human fibroblast monolayers. 125-I-EGF binds specifically to fibroblast monolayers. Half-maximal binding is observed at 4 times 10 minus 10 M EGF; at saturation of binding approximately 4 times 10-4 molecules of EGF are bound per cell. 125-I-Insulin is also bound specifically by intact monolayers with half-maximal binding observed at 10 minus 9 M insulin; about 4 times 10-3 molecules of insulin are bound per cell at saturation. Both insulin and EGF stimulate thymidine incorporation and alpha-aminoisobutyrate uptake. A half-maximal effect for insulin is observed at about 10 minus 9 M, both for the stimulation of thymidine incorporation and for the stimulation of alpha-aminoisobutyrate uptake; for EGF, half-maximal stimulation of both thymidine incorporation and alpha-aminoisobutyrate uptake is observed at 10 minus 10 M EGF. EGF causes an apparent greater stimulation of thymidine incorporation than does insulin, whereas the stimulation of alpha-aminoisobutyrate uptake is the same for both insulin and EGF. The degree of stimulation of alpha-aminoisobutyrate uptake by either insulin or EGF varied (1.2- to 2-fold) from one batch of cells to another, as did the measured values of the apparent K-m (average value 1 mM, range 0.6 to 2 mM) and V-max (average, 0.82, range 0.78 to 0.87 nmol/100 mug of protein per min) for alpha-aminoisobutyrate. Nonetheless, the apparent K-m of each peptide for stimulation of alpha-aminoisobutyrate uptake was independent of the degree of increase in alpha-aminoisobutyrate uptake, and was constant from one batch of cells to another. The peptide-mediated stimulation of alpha-aminoisobutyrate uptake can be attributed to a decrease in the apparent K-m for alpha-aminoisobutyrate (e.g. for insulin) from 0.70 to 0.57 mM; for EGF from 0.87 to 0.66 mM) and a concomitant increase in the apparent V-max for alpha-aminoisobutyrate (e.g. for insulin from 0.78 to 0.87 and for EGF from 0.80 to 0.84 nmol/min/100 mug of cell protein). The stimulation requires a 40- to 60-min period of preincubation with either peptide and is blocked by pretreating cells with cycloheximide. In the presence of ouabain, both peptides inhibit rather than stimulate alpha-aminoisobutyrate uptake; ouabain lowers the basal rate of alpha-aminoisobutyrate uptake. The uptake of 3-0-methyl-D-glucose is not affected by either EGF or insulin under conditions where insulin stimulates fat cell transport. These observations indicate that cultured human fibroblasts possess specific binding sites for insulin and EGF, which sites can be related to two actions of the peptides: stimulation of thymidine incorporation and alpha-aminoisobutyrate uptake.     

Human epidermal growth factor-on molecular forms present in urine and blood.

A sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of human epidermal growth factor (EGF) was employed to study EGF in urine and blood. The EGF/creatinine ratio in urine was significantly higher for women (range and (median); 0.20-0.83 (0.50) nmol EGF/mmol creatinine) than for men (0.17-0.63 (0.30) nmol EGF/mmol creatinine). We were not able to demonstrate EGF in plasma (median plasma EGF < 0.01 nmol/l) whereas serum contained a range and (median) of 0.02-0.31 (0.12) nmol EGF/l. The amount of EGF in serum showed a weak correlation to the platelet count (r = 0.327). EGF was partly purified by affinity chromatography from urine (urine EGF) and from activated platelets in platelet rich plasma (blood EGF). Both blood and urine contained a high molecular weight form of EGF (HMW EGF) as well as 6 kDa EGF. HMW EGF from blood was similar to HMW EGF from urine concerning behaviour upon gel filtration, pI and apparent affinity constant for binding to the EGF receptor. However, HMW EGF constituted approx. 40% of blood EGF but only 10% of urinary EGF. The 6 kDa EGF from both blood and urine contained two isopeptides with pI around 4.40 and 4.15 but in various proportions. The apparent affinity constant for binding to the EGF receptor for blood 6 kDa EGF was 1.8 x 10(10) l/mol compared to 1.0 x 10(10) l/mol for urinary 6 kDa EGF and 0.8 x 10(10) l/mol for HMW EGF from both blood and urine. The present study suggests that the processing of the EGF precursor differs in the blood and in the kidneys and that 6 kDa EGF from blood and urine binds to the EGF receptor with a higher apparent affinity constant than does HMW EGF.     

Altered secretion and processing of epidermal growth factor in adrenergic-induced growth of the rat submandibular gland

The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta-agonist), which caused up to a 400% increase in submandibular tissue weight after 3 weeks. The weight increase coincided with marked morphologic changes, with degranulation and an apparent decrement in the number of the GCT cells. Immunostaining against EGF revealed a reduction in the number of EGF-immunoreactive cells. Concomitantly, the glandular contents of 6-kDa EGF decreased from 12.86+/-3.42 nmol/gland (mean+/-S.E.M.) in controls to 0.26+/-0.03 nmol/gland. EGF mRNA levels, expressed relative to total RNA levels, only tended to be reduced after 3 weeks as judged from RT-PCR and in situ hybridization (ISH). The isoproterenol-treated rats had increased output of EGF in the saliva, but the salivary secretion of protein was also increased. In both glandular tissue and saliva, gel filtration revealed partially processed high molecular weight forms of EGF in the isoproterenol-treated rats. These data indicate that isoproterenol treatment leads to a hyperstimulatory state of the GCT cells, which then causes depletion of the cellular stores of mature EGF, and most likely due to a shortened posttranslational transit, incomplete peptide processing.     

Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.     

Renal uptake and excretion of epidermal growth factor from plasma in the rat

The rat excretes around 2 nmol epidermal growth factor (EGF) in the urine per 24 h. The urinary EGF might be derived from plasma and/or might be synthesized in the kidneys. We have used the rat to study the renal uptake and excretion of homologous EGF from plasma. I.v. injected 125I-EGF was removed from the circulation within a few minutes. 5 min after the injection, the kidneys contained 12% of the 125I-EGF. The kidneys seemed to degrade most of the 125I-EGF which they accumulated from blood, as only 4% of the injected label was excreted as intact 125I-EGF in the urine. The amount of endogenous EGF in plasma was under the detection limit of our enzyme-linked immunosorbent assay (0.03 nmol/l) and it remained so after bilateral nephrectomy. Even if plasma EGF was 0.03 nmol/l excretion of EGF from plasma could account for less than 5% of the urinary EGF. This study shows that the kidneys are able to accumulate EGF from plasma and excrete a part of it as intact EGF in the urine. However, excretion of immunoreactive EGF from plasma can only account for a minor part of the urinary EGF.     

Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Effect of sodium and osmolarity on renin secretion.

The effect of changes in sodium and osmolarity on renin secretion has been studied in the isolated perfused rat kidney. Perfusion with low sodium buffer (110 mM/l) produced a significant increase in renin secretion compared with control experiments (Na+:135 mM/l). Since the presence of tubules seems necessary for such an effect to take place, it suggests that the high renin secretion stimulated by a low sodium buffer centers in the Macula densa. Perfusion with high sodium buffer (170 mM/l; osmolarity 350 mOs/l) induces a stimulation on renin release. However, a greater rise in renin is achieved in control experiments if choline chloride increases the osmolarity from 300 to 350 mOs/l. All this suggests that high sodium buffer, independently of its osmotic effect, has an inhibitory role on renin release.     

Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration

We characterize herein the impact of myocardial nitric oxide (NO) synthesis on the inotropic response to two cardioactive peptides, endothelin-1 (ET-1) and adrenomedullin (AM). In the isolated perfused rat heart preparation, intracoronary infusion of AM (0.03 and 1 nmol/l) and ET-1 (0.08 and 1 nmol/l) for 30 min induced a dose-dependent, gradual increase in developed tension, the maximal responses being equal. Inhibition of myocardial NO synthase (NOS) by N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 micromol/l) enhanced the inotropic response to ET-1 at a concentration of 1 nmol/l; meanwhile, the effect of AM was not augmented significantly. The inotropic response to simultaneous administration of low, equipotent doses of AM (0.03 nmol/l) and ET-1 (0.08 nmol/l) was significantly smaller than that of either peptide alone. This depressed response was more than overcome by concomitant administration of L-NAME. In conclusion, this study reveals that the maximal inotropic response to ET-1 can be augmented by inhibition of myocardial NOS, whereas it has only a minor impact on the effect of AM. The inotropic response to combined administration of low doses of AM and ET-1 is substantially suppressed by endogenous NO, whereas the individual effects of the peptides at these doses are not the subject of secondary modulation by NO.     

The parotid gland is the main source of human salivary epidermal growth factor

To clarify the production of human epidermal growth factor (EGF) by different salivary glands, we measured its concentration by radioimmunoassay separately in whole saliva, in parotid gland (PG) saliva and in mixed submandibular (SMG) and sublingual gland (SLG) saliva. Also, we studied the presence of EGF in PG and SMG by immunohistochemistry. The mean (geometric) concentrations of EGF in PG saliva (2704 pg/ml, +/- SEM interval 2393-3056 pg/ml, n = 20) was higher (p less than 0.001) than in whole saliva (864 pg/ml, +/- 733-1019 pg/ml, n = 29), which in turn was higher (p less than 0.001) than in mixed SMG + SLG saliva (357 pg/ml, +/- 296-430 pg/ml, n = 16). No sex difference existed in any salivary gland EGF. Immunohistochemistry revealed EGF in the acinar cells of both PG and SMG, but only in PG there were prominent EGF deposits in luminal spaces. Our data suggest that EGF is produced by both PG and SMG, but that more of it is secreted from the PG. This result is new and challenges the general view that human salivary EGF is mainly from SMG. In mouse almost all salivary EGF comes from SMG and its amount is androgen dependent. Thus there are great differences in sources and regulation of salivary EGF between man and mouse.     

Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.     

Chemical characterization of the two forms of epidermal growth factor in murine saliva

Extracts of murine salivary glands contain two molecular forms of epidermal growth factor, EGF I and EGF II (Petrides, P.E., Levine, A.E., Shooter, E.M. in: Peptides: - Synthesis, Structure and Function (Rich, D.H., Gross, E.eds.) p. 781 (1981]. We have identified both molecules not only in salivary gland extracts but also in saliva using only reverse phase liquid chromatography methodology. EGF I and II were isolated from submaxillary gland extracts in a ratio of 3:1 regardless of whether the classical isolation procedure or our rapid RPLC based technique was used. Both molecular forms were present in the same ratio in saliva of mice of both sexes when salivation was induced by epinephrine treatment of the animals. As judged by amino acid analysis and N-terminal sequencing salivary EGF I corresponds to the 53 amino acid sequence of murine EGF and EGF II is Des-ASN-EGF. The observation that EGF and Des-ASN-EGF are consecreted into saliva upon epinephrine stimulation implies a physiological role of EGF II which may be related to the high molecular weight EGF-complex.     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

EGF receptor activities in mammalian development

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.     

Binding of insulin-like growth factor-I (IGF-I) to primary cultures of chondrocytes from rat rib growth cartilage

Binding of insulin-like growth factor I (IGF-I) to cultured resting, proliferative and hypertrophic growth plate chondrocytes was investigated. The optimal binding conditions and the extent of degradation of the 125I-IGF-I at 20 degrees C were analyzed in a time-course study. The maximal binding without noticeable degradation was observed after 3 h. The binding of IGF-I to the proliferative cells was 2-fold higher than to the resting and the hypertrophic cells. On the proliferative chondrocytes two classes of receptors with different affinities were found. 125I-IGF-I could be displaced from the proliferative cells by unlabelled IGF-I, IGF-II and insulin, respectively. Half maximal binding was observed at 0.3 nmol/l (= 2.2 micrograms/l) of IGF-I, 4.3 nmol/l (= 32 micrograms/l) of IGF-II and 350 nmol/l (= 2000 micrograms/l) of insulin. No specific binding of human growth hormone (hGH) could be demonstrated. When binding of epidermal growth factor (EGF) to the proliferative cells was assessed, little, but specific binding was observed.     

The activity of cathepsin D in saliva of cystic fibrosis patients

Cystic fibrosis (CF) is genetically determined illness, which is caused by the mutation in the CFTR gene. CFTR protein is also expressed in epithelial cells of parotid glands, therefore parotid glands are also affected in CF patients. Cathepsin D is one of the proteolitic cascade enzymes. Physiological wearing out result in occurrence of trace quantities of this enzyme in serum and body fluids, including saliva. Among different enzymes, saliva contains cathepsin D (CTSD, EC 3.4.23.5). The aim of this study was to determine cathepsin D activity in mixed saliva in cystic fibrosis patients and healthy controls. The study was performed in a group of 26 CF patients (10F, 16M). The results obtained in CF group was compared with the results of thirty healthy subjects (12F, 14M). From each subject 8 ml of mixed saliva was obtained: before and after the stimulation of saliva excretion using paraffin pledgets. Protein and glycoprotein content was assessed using Winzler's method. Protein concentration in controls and CF group before stimulation of excretion was 1.15+/-0.714 mg/mL and 1.54+/-0.925 mg/mL. After stimulation protein concentration in saliva has lowered to 0.88+/-0.77 mg/mL in CF group and 1.24+/-1.213 mg/mL in controls. Glycoprotein concentration in controls and in CF group was respectively: before stimulation 1.08+/-0.271 mg/mL and 1.05+/-0.344 mg/mL; after stimulation 0.92+/-0.292 mg/mL and 0.86+/-0.283 mg/mL. The activity of CTSD in controls was 45.9+/-24.98 Tyr nmol/mL/4h before stimulation and 109.3+/-56.94 Tyr nmol/mL/4h after stimulation of excretion. In CF group CTSD activity before stimulation was 134.5+/-81.80 Tyr nmol/mL/4h and after stimulation 134.4+/-62.18 Tyr nmol/mL/4h. Comparing the CTSD activity in both groups statistically significant difference has been revealed in samples collected before stimulation of excretion (p=0.013). The activity of cathepsin D in saliva of cystic fibrosis patient is significantly higher than in healthy controls before the stimulation of excretion with paraffin pledgets.     

Gastrin and epidermal growth factor induction of ornithine decarboxylase in rat colonic explants

An organ culture system was utilized to examine the effect of gastrin (G-17-I) and epidermal growth factor (EGF) on colonic mucosal ornithine decarboxylase (ODC) activity, and the expression of the ODC gene. Exposure of colonic mucosal explants to either gastrin or EGF (50-500 ng/ml) for only 4 h resulted in a profound stimulation (150-600%) in ODC activity over the basal level. These increases were essentially abolished by difluoromethylornithine (DFMO; 2 nmol/ml) or CaCl2 (2 umol/ml). Gastrin also activated the ODC gene in the colonic mucosa as evidenced by increased steady-state ODC mRNA levels in the colonic mucosal explants after 4 h exposure to the hormone, when compared with the controls. It is concluded that colonic mucosal ODC is responsive to both gastrin and EGF.     

Saturated fatty acids suppress adrenocorticotropic hormone (ACTH) release from rat anterior pituitary cells in vitro

We studied whether fatty acids modify adrenocorticotropic hormone (ACTH) release induced by stimulation with corticotropin-releasing hormone (CRH) from rat anterior pituitary cells. Stimulation with CRH (0.01-100 nmol/l) significantly and concentration-dependently increased ACTH release, which was synergistically enhanced by the simultaneous stimulation with 1 nmol/l arginine-vasopressin. Addition of saturated fatty acids (butyrate, caprylate, laurate, palmitate and stearate) in a medium at 1 mmol/l, despite effects on the basal release, significantly reduced the ACTH release induced by CRH (1 nmol/l) stimulation. Caprylate suppressed ACTH release in a concentration-dependent manner. However, unsaturated C18 and C20 fatty acids (oleate, linolate, linolenate and arachidonate) at 1 mmol/l significantly increased the basal release, but none of them suppressed CRH (1 nmol/l)-induced ACTH release. In the presence of caprylate (1 mmol/l), CRH (1 nmol/l)-stimulated increase in cellular calcium ion concentration was diminished. From these results we conclude that saturated fatty acids have a suppressing effect on CRH-induced ACTH increase in primary cultured rat anterior pituitary cells.