Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells

Activins, initially identified as FSH-releasing proteins, have now been shown to exert effects on other cell types of the anterior pituitary, including the somatotrophs. In the present study the inhibitory action of activin-A (beta A beta A) on GH secretion was characterized using primary cultures of rat anterior pituitary cells. Activin-A suppressed basal GH secretion for up to 72 h (the longest time tested). Immediately after the treatment period with activin-A, when the cells were thoroughly washed and further incubated with or without rat GH-releasing factor (rGRF), basal and stimulated GH secretion were partially inhibited as well. In parallel, activin-A pretreatment diminished rGRF-stimulated cAMP accumulation. The effects of activin-A were time- and concentration-dependent, with half-maximal inhibition occurring in the range of 20-30 pM activin-A. A minimum pretreatment time of 3 h was required for maximal effect, and when rGRF and activin-A were added simultaneously, no inhibition was evident. Secretory responses of activin-A-pretreated cells to rGRF were influenced by glucocorticoids. When cells were cultured in the presence of the synthetic glucocorticoid dexamethasone, pretreatment (72 h) with activin-A attenuated rGRF-stimulated GH secretion only during short (1-h), but not longer (3-h), exposure periods to the neuropeptide. In the absence of dexamethasone, rGRF-stimulated GH secretion was inhibited at all incubation times tested (up to 3 h). A 3-h exposure to the protein factor did not alter total (cellular plus secreted) immunoreactive GH levels, suggesting that the inhibition of secretion with the shorter treatment was not secondary to attenuated GH biosynthesis. However, longer (72-h) treatment with activin-A decreased total GH levels, indicating lower GH biosynthetic rates, as previously shown. Somatostatin is recognized as the primary negative modulator of GH secretion. Activin-A and SRIF inhibited GH secretion additively, suggesting distinct mechanisms of action for each. GH secretion in response to other secretagogues, such as 12-O-tetradecanoyl-phorbol-13-acetate, forskolin, cholera toxin, and 8-bromo-cAMP, was also suppressed after activin-A pretreatment. The presence of the RNA synthesis inhibitor actinomycin-D completely blocked the inhibitory effect of a 3-h activin-A pretreatment on subsequent rGRF-stimulated GH secretion. Pertussis toxin was only partially effective in preventing the inhibition by activin-A. The results of this study indicate that activin-A plays a crucial role as a modulator of somatotropic function, inhibiting GH secretion at the level of the secretory process and secondary to the inhibition of GH biosynthesis.     

Growth hormone secretion by cultured rat anterior pituitary cells. Effects of culture conditions and dexamethasone

Optimal conditions were sought for the study of GH secretion by cultured normal pituitary cells. Dispersed rat pituitary cells were cultured for 1 week in four different media supplemented with 10% fetal calf serum. Minimal essential medium resulted in high GH content and secretion during a 4-h incubation period, whereas GH secretion was lower (P less than 0.05) for cells cultured in medium 199, Ham's F-10, and RPMI-1640. GH secretion/24 h declined gradually with time. After 2 weeks in culture hormone secretion amounted to 30% of secretion on day 1, but after 3 weeks GH secretion was still measurable. GH recovery during the 3-weeks culture period was more than 600% of the amount initially plated. GH secretion was positively correlated with the bicarbonate concentration between 0.85 and 2.2 g/liter NaHCO3. When pituitary cells were cultured in concentrations varying from 0.5 X 10(5) to 10 X 10(5) cells per dish, GH secretion and content per cell were constant, suggesting that no direct autofeedback occurred in cultures with high cell densities and thus high medium GH. Dexamethasone stimulated GH secretion and content in a dose-dependent way (0.1 nM-10 microM). The stimulatory effect of 100 nM dexamethasone occurred within 24-48 h. After 7 days of treatment with 100 nM dexamethasone, GH secretion had increased to 190% and GH content to 230% of control. In contrast to the effects on GH, dexamethasone suppressed PRL secretion in a dose-dependent way, but this effect was seen only after 7 days of treatment and not after 4 days of treatment. Cycloheximide and actinomycin D prevented the stimulatory effect of dexamethasone on GH secretion. However, 24 h after cessation of cycloheximide treatment GH secretion was stimulated by dexamethasone. Four days of treatment with 100 nM dexamethasone did not affect the GH response to somatostatin, prostaglandin E1, and theophylline, nor the PRL response to dopamine, TRH, and theophylline. Thus, culture conditions may affect GH production, and dexamethasone can be used to culture somatotrophs for longer periods with steady GH production and normal responsiveness.     

The interrelationship between the effects of insulin-like growth factor I and somatostatin on growth hormone secretion by normal rat pituitary cells: the role of glucocorticoids

Both insulin-like growth factor I (IGF-I) and somatostatin (SRIH) have been shown to directly inhibit GH release and the total GH content of cultured pituitary cells. In the present study we evaluated the interrelationship between the effects of a recombinant human IGF-I analog ([Thr59]IGF-I) and SRIH on GH release by cultured normal rat pituitary cells together with the effects of glucocorticoids. In all experiments anterior pituitary cells were preincubated for 24 h without or with IGF-I, SRIH, and/or dexamethasone. Thereafter, 24-h incubations without or with IGF-I, dexamethasone, SRIH, and GHRH were performed. Both IGF-I and SRIH inhibited basal and GHRH-stimulated GH release in a dose-dependent manner; the maximal inhibitory concentrations were 5 nM IGF-I and 10 nM SRIH. These concentrations inhibited basal and GHRH-stimulated GH release by 23% and 40% (IGF-I) and 80% and 85% (SRIH), respectively. The combination of IGF-I and low concentrations of SRIH exerted an additive inhibitory effect on GHRH-stimulated GH release; IGF-I (1 nM) and SRIH (10 pM) together inhibited GH release by 50%, while the maximal inhibitory concentrations of 5 nM IGF-I and 10 nM SRIH virtually completely inhibited GH release (by 93%). Preincubation with 5 and 100 nM dexamethasone attenuated the sensitivity of somatotrophs to SRIH and completely abolished the inhibitory effects of IGF-I. This effect of dexamethasone could be reversed by coincubation with the glucocorticoid receptor antagonist RU 38486. High concentrations of 5-10 nM of the recombinant human IGF-I analog stimulated PRL cell content (5 and 10 nM) and release (10 nM), while a purified IGF-I preparation extracted from human blood exerted a parallel inhibitory effect on GH and PRL release. We conclude that 1) IGF-I and SRIH exert an additive direct inhibitory effect on basal and GHRH-stimulated GH secretion by normal cultured pituitary cells; 2) glucocorticoids directly attenuate the sensitivity of somatotrophs to SRIH, but completely prevent the inhibitory effects of IGF-I on GH secretion; and 3) in contrast to a purified IGF-I preparation extracted from human blood (which inhibits GH and PRL release) high concentrations of the recombinant IGF-I preparation (which inhibit GH release) stimulate PRL production.     

Regulation of hormone release by cultured cells from a thyrotropin-growth hormone-secreting pituitary tumor. Direct inhibiting effects of 3,5,3'-triiodothyronine and dexamethasone on thyrotropin secretion

The regulation of TSH and GH secretion was investigated in cultured tumor cells prepared from a mixed TSH/GH secreting pituitary tumor. The tumor tissue had been removed transsphenoidally from a patient with hyperthyroidism and inappropriately high serum TSH levels and acromegaly. TSH and GH secretion by cultured cells were stimulated in a parallel way by TRH (300 nM) and LHRH (50 nM), but were unaffected by bromocriptine (10 nM). Exposure of the tumor cells to dexamethasone (0.1 microM) or T3 (50 nM) had differential effects on hormone secretion. GH secretion was greatly stimulated by dexamethasone, but unaffected by T3. TSH secretion was inhibited both by T3 and by dexamethasone. So, T3 and glucocorticoids inhibit TSH release by the human pituitary tumor cells studied at least partly by means of a direct effect.     

Glucocorticoids: potent inhibitors and stimulators of growth hormone secretion

Excessive glucocorticoid concentrations are well recognized inhibitors of linear growth, due in part to their suppression of GH secretion. The mechanism of this inhibition has been unclear, especially since glucocorticoids enhance the in vitro GH response of pituitary cells to GH-releasing hormone (GHRH). We investigated the possibility that hypothalamic somatostatin might be mediating these dichotomous observations by using passive immunization techniques. The GH response to GHRH was significantly blunted in rats pretreated with the synthetic glucocorticoid, dexamethasone, compared to that in normal animals. In marked contrast, the immunoneutralization of somatostatin resulted in a significantly enhanced GH response to GHRH in dexamethasone-treated animals. These results suggest that the previously described inhibitory action of glucocorticoids on GH secretion in vivo are mediated via altered hypothalamic somatostatin tone.     

On the role of the peptide galanin in regulation of growth hormone secretion.

The effects of the peptide galanin on growth hormone secretion were studied in vitro using cultured rat and human anterior pituitary cells, and in vivo by iv administration of galanin in both rats and humans. Galanin in concentrations from 10 nmol/l to 1 mumol/l did not alter basal GH release, but slightly inhibited GHRH-stimulated GH release from cultured rat anterior pituitary cells. Galanin (1 mumol/l) did not significantly change basal or GHRH-stimulated GH secretion from cultured human anterior pituitary cells. In contrast, iv injection of 1 microgram (300 pmol) galanin to rats induced an increase in plasma GH that was reproducible at repetitive injections. The galanin-induced GH release in rats was of a lower magnitude than the increase in plasma GH after iv injections of GHRH, and was seen with a 5-15 min delay in comparison to iv administered GHRH. In man, iv infusions of galanin (40 pmol.kg-1.min-1.(40 min)) also caused a significant increase in plasma GH, but it occurred 25-30 min after the beginning of the infusion. These results suggest an indirect action of galanin on GH release in both rats and humans, i.e. galanin does not directly affect the somatotropes. In agreement with a central action, no binding sites for galanin could be demonstrated in the rat anterior pituitary by autoradiography. Since galanin did not affect somatostatin release from fragments of rat mediobasal hypothalamus, the stimulatory effects of galanin on GH release are most likely mediated via a stimulatory effect on GHRH neurons.     

Identification of the somatostatin receptor subtypes (sst) mediating the divergent, stimulatory/inhibitory actions of somatostatin on growth hormone secretion

It is well established that somatostatin acts through G protein-coupled receptors, termed sst, to inhibit GH release. However in pigs somatostatin can stimulate or inhibit in vitro GH secretion in a dose- and somatotrope subpopulation-dependent manner. We report herein that somatostatin-stimulated GH release is blocked by pretreatment with GTPgamma-S, suggesting an involvement of G protein-coupled receptors. Consistent with this, an sst5 selective agonist stimulated spontaneous GH secretion at doses ranging 10(-13) to 10(-9) m, without influencing GHRH-induced GH release. Conversely, sst1-, sst2-, sst3-, and sst4-specific agonists inhibited GHRH-evoked GH release but not basal GH secretion. Examination of the effects of sst-specific agonists on two subpopulations of somatotrope cells separated by density gradient centrifugation [low- (LD) and high-density (HD) cells] showed that only a low dose of the sst5 agonist stimulated GH release in LD somatotropes, whereas both low and high doses of this agonist stimulated GH release in HD cells. In marked contrast, sst1 and sst2 agonists blocked GHRH-stimulated GH release in LD cells at all doses tested, whereas only a high dose of the sst2 agonist inhibited GHRH-induced GH release in HD somatotropes. Interestingly, sst expression pattern in these subpopulations correlates with the distinct actions of sst-selective agonists; specifically, sst5 is more abundant in HD somatotropes, whereas sst1 and sst2 mRNA predominate in LD cells. These results indicate that in the pig, sst1 and sst2 are the primary mediators of the inhibitory effects of somatostatin, whereas sst5 or an sst5-related mechanism mediates the stimulatory action of somatostatin on GH release.     

Cellular mechanism of caprylic acid-induced growth hormone suppression

To determine the dynamic secretory pattern of growth hormone (GH) in the presence of free fatty acids (FFA), we studied the effect of caprylic acid on basal and rGRF(1-29)NH2 (GRF)-induced GH secretion in acutely dispersed and perifused rat pituitary cells. At a concentration of 3.0 mmol/L, caprylic acid inhibited both basal (P less than .05) and GRF-stimulated GH secretions (P less than .01), except when the maximal (near the EC100) GRF concentration of 100 pmol/L was used. Lower concentrations of caprylic acid such as 0.3 and 1.0 mmol/L significantly inhibited, in a concentration-dependent manner, GH secretion induced by a 6.25-pmol/L GRF. However, at a GRF concentration of 25 pmol/L, this inhibitory effect was abolished. The time-course of GH response to GRF was similar in both control and caprylic acid-treated cells. To elucidate the mechanism(s) of action of the caprylic acid-induced blockade of GH secretion, in 3-day cultured rat pituitary cells, the effect of caprylic acid on basal and GRF-stimulated GH and 3',5'-cyclic adenosine monophosphate (cAMP) release. We also tested its effect on the Ca2+ ionophore, A23187-induced GH release. Caprylic acid (0.3 to 3.0 mmol/L) significantly inhibited basal GH release and GRF- or A23187-induced GH secretion. Furthermore, it decreased both basal and GRF-stimulated cAMP release (P less than .05). In addition, the effect of caprylic acid on rGRF(1-29)NH2 affinity to GRF pituitary binding sites was determined using [125I-Tyr10]hGRF(1-44)NH2 as radioligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of growth hormone receptors in primary cultured rat hepatocytes

The hormonal regulation of GH receptors was studied by measuring specific binding of [125I]human GH to primary cultured rat hepatocytes. The binding of labeled GH to primary cultured hepatocytes decreased during culture, but addition of dexamethasone (100 nM) compensated for this decrease and even increased GH binding. After addition of dexamethasone, the binding increased to a maximum after 10 h, and after 24 h was about 6 times that of control cells. Glucagon (100 nM) did not have any significant effect on GH binding by itself, but enhanced the increased binding caused by dexamethasone about 1.5-fold. For this effect, glucagon could be replaced by (Bu)2cAMP. Insulin (10 nM) and epidermal growth factor (20 ng/ml) reduced the increase by dexamethasone plus glucagon by about half. Scatchard plot analysis showed that the changes of GH binding induced by various hormones were due to changes in the number of binding sites without significant changes in their affinity. The GH bound to dexamethasone or dexamethasone plus glucagon-treated cells was not replaced by unlabeled ovine PRL. This strongly suggests that the number of somatogenic (GH) receptors may be subject to hormonal regulation: dexamethasone alone or with glucagon may induce GH receptors, whereas insulin and EGF may suppress the induction of GH receptors. These patterns of hormonal regulations were almost the same as those of proteins whose expressions were known to be differentiated functions of liver. On the other hand, the increase of GH binding by dexamethasone was inhibited by cycloheximide and actinomycin D, though the GH binding was inhibited by cycloheximide, but not by actinomycin D in the cells cultured without dexamethasone. This result suggests that the increased binding induced by dexamethasone is dependent on the synthesis of new protein and is probably regulated at a pretranslational level.     

Stimulation and inhibition of pituitary growth hormone release by angiotensin II in vitro

Rat pituitary cell aggregates cultured in serum-free chemically defined medium, single cells, and hemipituitaries were used in a perifusion system to study the influence of angiotensin II (AII) on GH release. In aggregates the peptide displayed both stimulatory and inhibitory effects on GH release, depending on the hormonal conditions of the culture medium and the age of the animal. When cultured in the absence of glucocorticoid, a modest but statistically significant stimulation was seen in aggregates from immature as well as adult animals. In aggregates from 5-day-old animals, dexamethasone (DEX) strongly enhanced the GH-releasing activity of AII in a dose-dependent way; in aggregates from 14- and 25-day-old rats, the same pattern was found, although the stimulatory action was weaker than the effect in 5-day-old rats. In aggregates from adult animals, the glucocorticoid established an inhibitory effect of AII on GH release, an effect seen with both low and high concentrations of DEX. These age- and DEX-dependent effects were not found for AII stimulation of PRL release. In the presence of DEX, AII also inhibited GRF-induced GH release in aggregates from adult animals, while it was synergistic with GRF in aggregates from developing animals. The effects of AII on GH release disappeared when aggregates were redispersed into single cells. However, in these single cell preparations AII strongly stimulated PRL release. In hemipituitaries from 1-, 5-, and 14-day-old animals, AII also stimulated GH release, but no effect was seen in hemipituitaries from 25-day-old and adult animals. These data indicate that AII has dual effects on GH release depending on the developmental stage of the animal and the hormonal environment. Furthermore, since no effect of AII was seen after redispersion of aggregates into single cells, both stimulatory and inhibitory effects seem to be based on an intercellular signaling system.     

Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors.

GH secretion has been thought traditionally to be regulated by the two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF). Recent evidence has suggested that other factors may be involved. These factors include the natural ligand for the synthetic hexapeptide GH-releasing peptide (GHRP) and the putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PA-CAP). Accordingly, we examined the effects of GHRP and PACAP on GH secretion at the single cell level using the reverse hemolytic plaque assay which allows distinction of effects on the number of secreting cells and the amount of hormone each cell secretes. Both factors stimulated GH secretion in a dose-dependent fashion, with PACAP being more effective. PACAP increased both the number of cells secreting and the mean amount of hormone secreted per cell. In contrast, GHRP increased the number of secreting cells, although it had no effect on the amount of secretion per cell. GH secretion induced by GHRH, GHRP, and PACAP was inhibited by SRIF, but the effect was predominantly on the number of cells secreting rather than the amount secreted per cell. Specific antagonists to GHRP and GHRH inhibited GH secretion induced by the respective agonist but not that induced by the other factor nor by PACAP. These findings confirm the complex nature of the regulation of GH secretion at the level of the somatotrope. At least three factors, operating via distinct receptors, are able to increase GH secretion. In addition, they ascribe a potential physiological role for the hitherto putative hypophysiotropic factor PACAP.     

Insulin regulation of rat growth hormone gene expression

Insulin has been shown previously to inhibit basal and glucocorticoid- or T3-stimulated rat GH (rGH) synthesis, secretion, and mRNA levels in cultured rat pituitary tumor cells (GH3 cells) or pituitaries. The effects of insulin on rGH gene expression in GH3 cells were examined in greater detail in the current studies. Cells were deinduced for 5 days in medium devoid of steroids, T3, and insulin. Cells were then treated for 48 h with insulin (5 X 10(-9) M), dexamethasone (Dex; 10(-6) M), T3 (10(-8) M), insulin plus Dex, or insulin plus T3. When media and hormones were not replaced daily the results were similar to those obtained previously. Insulin decreased both basal and glucocorticoid-stimulated rGH mRNA levels to approximately 70% of control levels, as measured by cytoplasmic dot hybridization. By contrast, when media and hormones were replaced daily, rGH mRNA levels increased by 1.5 to 7-fold in response to insulin in the absence or presence of Dex or T3, measured by both cytoplasmic dot hybridization and RNA (Northern) blotting. Dex increased rGH mRNA levels under both sets of conditions, verifying the specific nature of the insulin influence. Maximum rGH gene expression was achieved after a 48-h exposure to insulin. The observed insulin effects were probably mediated through insulin rather than insulin-like growth factor I or II receptors, since the concentration of insulin employed was near the Kd of the hormone for its receptor measured in the same cells. These results suggest that insulin is capable of regulating rGH gene expression. The action of insulin can be either positive or negative and is influenced by the metabolic state of the cell.     

Pituitary adenylate cyclase-activating polypeptide, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells

There is increasing evidence that hormones play an important role in the control of endothelial cell function and growth by regulating the production of vascular endothelial growth factor (VEGF). VEGF regulates vascular permeability and represents the most powerful growth factor for endothelial cells. In the normal anterior pituitary, VEGF has been detected only in folliculostellate (FS) cells. In the present study, the regulation of the release of VEGF from FS-like mouse TtT/GF cells, and from FS cells of rat pituitary monolayer cell cultures was investigated using a specific VEGF ELISA. Basal release of VEGF was demonstrated in cultures of both TtT/GF cells and rat pituitary cells. Interestingly, the VEGF secretion was stimulated by both forms of pituitary adenylate cyclase-activating polypeptide (PACAP-38 and PACAP-27), indicating that this hypothalamic peptide regulates endothelial cell function and growth within the pituitary. VEGF secretion was also stimulated by interleukin-6 (IL-6) whereas basal, IL-6- and PACAP-stimulated secretion was inhibited by the synthetic glucocorticoid dexamethasone. The inhibitory action of dexamethasone was reversed by the glucocorticoid receptor antagonist RU486, suggesting that in FS cells functional glucocorticoid receptors mediate the inhibitory action of glucocorticoids on the VEGF secretion. The endocrine and auto-/paracrine control of VEGF production in pituitary FS cells by PACAP, IL-6 and glucocorticoids may play an important role both in angiogenesis and vascular permeability regulation within the pituitary under physiological and pathophysiological conditions.     

Growth hormone secretion from chicken adenohypophyseal cells in primary culture: effects of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release

A primary culture of chicken adenohypophyseal cells has been developed to study the regulation of growth hormone (GH) secretion. Following collagenase dispersion, cells were exposed for 2 hr to vehicle (control) or test agents. Human pancreatic (tumor) growth hormone-releasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor stimulated GH release to similar levels. GH release was increased by the presence of dibutyryl cyclic AMP. Thyrotropin-releasing hormone (TRH) alone did not influence GH release; however, TRH plus hpGRF together exerted a synergistic (greater than additive) effect, increasing GH release by 100 to 300% over the sum of the values for each secretagogue acting alone. These relationships between TRH and hpGRF were further examined in cultured cells exposed to secretagogues for two consecutive 2-hr incubations. TRH pretreatment enhanced subsequent hpGRF-stimulated GH release by about 80% over that obtained if no secretagogue was present during the first incubation. In other experiments, somatostatin (SRIF) alone did not alter GH secretion. However, SRIF reduced hpGRF-stimulated GH release to levels found in controls. Furthermore, GH release stimulated by the presence of both TRH and hpGRF was lowered to control values by SRIF. The results of these studies demonstrate that a primary culture of chicken adenohypophyseal cells is a useful model for the study of GH secretion. Indeed, these results suggest that TRH and hpGRF regulate GH secretion by mechanisms which are not identical.     

Involvement of corticotropin-releasing factor and somatostatin in stress-induced inhibition of growth hormone secretion in the rat

Corticotropin-releasing factor (CRF), which is released into the pituitary portal blood during exposure to noxious stimuli, can act centrally to inhibit GH secretion. We have investigated a possible role of endogenous CRF in mediating the stress-induced inhibition of GH release in the rat. While exposure to electroshocks markedly lowered plasma GH levels measured 20 min later, the central administration of the CRF antagonist alpha-Hel CRF-(9-41) totally abolished the effect of stress. To examine possible mechanisms through which CRF might mediate the inhibitory action of various stimuli on GH secretion, we have administered antisomatostatin (anti-SS) serum to CRF-injected rats and observed that immunoneutralization of endogenous SS blocked the inhibitory action of CRF on basal plasma GH values. Additionally, we have shown that CRF acted centrally to prevent the stimulatory action of both exogenously administered GH-releasing factor and the endogenous GH-releasing factor released by morphine sulfate. These effects were abolished by previous treatment with anti-SS serum. Such observations support the hypothesis that in the rat, endogenous CRF mediates the inhibitory action of noxious stimuli on GH secretion and further suggest that this effect may involve an increased release of endogenous SS.     

The growth hormone (GH)-releasing hormone (GHRH)-GH-somatomedin axis: evidence for rapid inhibition of GHRH-elicited GH release by insulin-like growth factors I and II

Hypothalamic-pituitary-end-organ axes are frequently controlled by long loop negative feedback homeostatic mechanisms. Insulin-like growth factor I (IGF-I), IGF-II, and insulin receptors have recently been described in normal and neoplastic rat and acromegalic human pituitary cells, a finding which suggests the possibility that somatomedins might exert feedback at the level of the anterior pituitary. To study the kinetics of this feedback response, we used perifused dispersed rat anterior pituitary cells to learn if somatomedins or insulin could inhibit GH-releasing hormone (GHRH)-stimulated GH secretion. Cells were exposed to hourly boluses of 1 nM GHRH with or without varying doses of IGF or insulin. IGF-I inhibited GHRH-elicited GH release with an IC50 of 6.5 nM; maximal inhibition (approximately 67%) was achieved with 10 nM IGF-I. IGF-II was a less potent hormone, with 10 nM inhibiting about 30% of GHRH-stimulated GH release. Slight inhibition of stimulated GH release (less than 15%) was seen when cells were treated with insulin, but only when doses of insulin of 10 nM or more were used. In conclusion, nanomolar concentrations of IGF-I and IGF-II inhibited GHRH-elicited GH release from perifused rat pituitary cells in a dose-dependent manner; and insulin was not an effective inhibitor of stimulated GH release at physiological peptide concentrations. In conjunction with our previous findings that the concentrations of IGF-I and IGF-II receptors greatly exceed that of insulin receptors on normal rat pituitary cells, we hypothesize that the GH-inhibiting action of high dose insulin is mediated through an IGF receptor.