Endocrinology

This study tested the hypothesis that the fetal llama, a species adapted to the chronic hypoxia of life at high altitude, demonstrates a potent carotid chemoreflex influence on adrenocortical responses during acute hypoxemia. Plasma ACTH and cortisol concentrations, and mesencephalic and adrenal blood flows were measured during a 1-h period of acute hypoxemia in six intact and four carotid sinus-denervated llama fetuses at 0.6-0.7 of gestation. Fetal PaO2 was reduced from approximately 23 to about 14 mm Hg in both intact and carotid-denervated groups during acute hypoxemia. During hypoxemia, fetal plasma ACTH, adrenal blood flow, and, therefore, delivery of ACTH to the adrenals increased to similar extents in both intact and carotid-denervated fetal llamas. Despite this, the increase in plasma cortisol in hypoxemia in intact fetuses was absent in carotid-denervated fetuses. In addition, the increase in delivery of cortisol to the mesencephalon calculated in intact fetuses during hypoxemia did not occur in the carotid-denervated group. These data suggest that the integrity of the carotid chemoreceptors is indispensable to cortisol release during acute hypoxemia in the llama fetus, even at 0.6-0.7 of gestation.

Androstenedione treatment of pregnant monkeys at 0.8 of gestation reproduces endocrine, biophysical, and biochemical changes similar to those measured during spontaneous, term labor in the pregnant monkey. In the pregnant baboon, the spontaneous onset of labor at term has been attributed to a forward shift in the nocturnal estradiol surge relative to that of progesterone in maternal plasma. This study investigated whether androstenedione treatment of the pregnant baboon at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal surge of maternal plasma estradiol relative to progesterone and whether this shift is associated with premature increases in nocturnal myometrial activity. Eight pregnant baboons were prepared surgically under general anesthesia with vascular catheters and myometrial electromyogram electrodes between 121 and 139 days of gestation (term is ca. 185 days). Catheters were maintained patent by continuous infusion of heparinized saline from the time of surgery until one of two treatments began following at least 9 days of postoperative recovery. In four baboons (Group I), the saline administration was replaced by a continuous infusion of 10% intralipid vehicle during Day 1 of the experimental protocol. During Day 2 and Day 3, the intralipid infusion was switched for a continuous infusion of androstenedione dissolved in intralipid set at a low (0.8 mg x kg(-1) x h(-1)) and at a high (1.6 mg x kg(-1) x h(-1)) dose, each delivered for 24 h. The other four pregnant baboons (Group II) received 10% intralipid vehicle for Days 1, 2, and 3 of the experimental protocol. One baboon from Group I received an additional dose of 0.4 mg x kg(-1) x h(-1) for 24 h before the low and the high dose of androstenedione. In each baboon, during each experimental day, maternal arterial blood samples (1 ml) were taken at 1 h intervals for 12 h, starting 3 h before the onset of darkness in the animal's environment, for measurement of maternal plasma estradiol and progesterone concentrations via RIA. Myometrial contractions were counted during each night-time period of the experimental protocol. All pregnant baboons demonstrated increases in maternal plasma estradiol and progesterone concentrations at night-time. Androstenedione had a dose-dependent effect in elevating day-time maternal plasma estradiol concentrations and in promoting a forward shift in the nocturnal surge of maternal plasma estradiol without affecting the nocturnal progesterone profile in maternal plasma. Maternal treatment with androstenedione also led to an increase in nocturnal myometrial contraction activity. We conclude that androstenedione treatment of the pregnant baboon at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal estradiol surge relative to that of progesterone in maternal plasma and that this shift is associated with an increase in nocturnal myometrial contraction activity, in a similar way to that measured during spontaneous onset of labor at term in this species.

To ascertain if reductions in fetal plasma cortisol cause increases in fetal plasma ACTH, we treated pregnant ewes or their fetuses with aminoglutethimide (10 mg/kg BW) and metyrapone (20 mg/kg BW) and measured the hormonal responses with RIAs. When given to fetuses (n = 9) at 0.90 +/- 0.01 gestation (term-145 days), the steroid synthesis inhibitors reduced fetal plasma cortisol from 35.1 +/- 11.9 to 18.5 +/- 6.2 ng/ml (P less than 0.01) and plasma ACTH increased from 37 +/- 7 to 189 +/- 74 pg/ml (P less than 0.02). Thus, late in gestation cortisol from the fetal adrenal suppresses basal fetal ACTH secretion. Blockade of steroid biosynthesis in pregnant ewes carrying intact fetuses at 0.76 +/- 0.02 gestation (n = 11) or adrenalectomized fetuses at 0.81 +/- 0.01 gestation (n = 6) also reduced cortisol and increased ACTH in fetal plasma. In intact fetuses cortisol declined from 9.4 +/- 2.0 to 3.6 +/- 0.9 ng/ml (P less than 0.05), and ACTH increased from 46 +/- 8 to 183 +/- 67 (P less than 0.01); cortisol declined in adrenalectomized fetuses from 2.1 +/- 0.4 to 1.1 +/- 0.3 ng/ml (P less than 0.01), and ACTH increased from 106 +/- 13 to 400 +/- 104 pg/ml (P less than 0.01). Cortisol infusions into intact and adrenalectomized fetuses prevented both the decline in steroid concentration caused by the biosynthesis inhibitors given to the ewe and the increase in fetal plasma ACTH concentration. These data indicate that reductions in plasma cortisol in adrenalectomized fetuses or intact fetuses at a time in development when the fetal adrenal produces little cortisol cause compensatory increases in fetal plasma ACTH concentration. The simplest explanation for these observations is that from approximately 0.70 gestation, basal fetal ACTH secretion is tonically inhibited by cortisol circulating in fetal plasma. This cortisol can originate from sources other than the fetal adrenal.

Insulin modifies the effects of PTH on osteoblast-like cells. However, the basis for this effect is unknown. In bone and kidney cells, the effects of PTH on cellular function are mediated by second messengers generated through both the phospholipase C and adenylate cyclase systems. Therefore, we examined the effects of insulin on PTH second messenger generation in UMR-106-01 rat osteoblastic osteosarcoma cells. PTH produced a rapid, transient increase in intracellular free calcium concentration ([Ca2+]i) which was maximal at 30 sec and was only minimally reduced in the absence of extracellular calcium. Inositol-triphosphate (IP3) production was increased in parallel. PTH stimulation of [Ca2+]i was concentration-dependent from 0.5-1,000 nM, with half-maximal stimulation at approximately 50 nM PTH. A 30-sec exposure to 50 nM PTH produced 32% and 23% increases in IP1 and IP3 production, respectively (both P less than 0.05). Although insulin alone did not significantly alter basal [Ca2+]i, a 1-min exposure to 1-100 nM insulin produced a concentration-dependent suppression of the PTH-stimulated transient increase in [Ca2+]i and IP3 generation. 100 nM insulin decreased 50 nM PTH stimulation of [Ca2+]i and IP3 levels by 84% (P less than 0.02) and 80% (P less than 0.001), respectively. Preexposure to insulin also decreased PTH stimulation of intracellular cAMP levels, but to a lesser degree. A 1-min exposure to 100 nM insulin produced a 32% (P less than 0.01) decrease in PTH-stimulated cAMP generation, but lower insulin concentrations were without significant effects. These results demonstrate that in UMR-106-01 cells, insulin suppresses PTH stimulation of second messengers generated through both the phospholipase C and adenylate cyclase systems, but has a more marked effect on the former.

Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.

The accumulation of iron or aluminum can cause metabolic bone disease, but the mechanisms by which these agents affect bone metabolism remain uncertain. Since transferrin (Tf) can bind several different metals in plasma, equilibrium radioligand binding studies were performed to identify and characterize the Tf receptor in UMR-106-01 osteoblast-like cells; the role of Tf as a modifier of metal-induced changes in cell proliferation was also examined. Osteoblast-like cells grown in serum-free medium have approximately 40,000 Tf receptors on the cell membrane. Tf receptor expression increases during iron depletion and decreases with iron supplementation; the number of Tf receptors was also inversely related to both cell density and the rate of cell proliferation in vitro. Physiological levels of unsaturated Tf (5 microM) enhanced DNA synthesis in osteoblast-like cells maintained in serum-free medium, as measured by the incorporation of tritiated thymidine into DNA. Although neither 10 microM iron (Fe) nor 10 microM gallium (Ga), a known antiproliferative agent, altered DNA synthesis in UMR-106-01 cells during 48-h incubations in serum-free medium, both agents reduced the rate of DNA synthesis when added to serum-free medium containing 5 microM apo-Tf. Decreases in the incorporation of [3H] thymidine into DNA were also noted in osteoblast-like cells incubated for 48 h with 3 microM partially saturated iron Tf or gallium Tf. The results indicate that osteoblast-like cells have a single class of membrane receptors for Tf and that the regulation of Tf receptor expression in UMR-106-01 cells is similar to that in other cell types. The uptake of iron and gallium via the Tf-receptor complex can affect osteoblast proliferation, and such a mechanism may contribute to the bone cell toxicity of various metals.

The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.

PTH administration in vivo increases osteoblast number and activity, resulting in increased bone formation, and also increases osteoclast-mediated bone resorption. Studies in vitro, however, have shown that the actions of PTH on osteoblast-like cells are inhibitory and catabolic, as shown by decreases in growth rate and collagen synthesis and increases in collagenase production. The present studies were designed to investigate possible mechanisms for these observations by examining the effects of PTH on the response of osteoblast-like cells to the osteoblast growth factor, epidermal growth factor (EGF). Confluent cultures of UMR 106-01 cells were treated with rat PTH-(1-34) for periods up to 72 h, and EGF receptors were measured with [125I]EGF. PTH, in a dose- and time-dependent manner, increased the number of EGF receptors 2-fold. The half-maximal effect of PTH occurred at a concentration of 1 nM, the same PTH concentration that resulted in half-maximal increases in cAMP generation. The increase in EGF binding was associated with an enhanced biological effect, as shown by augmentation of EGF-stimulated diglyceride production. The effect of PTH could be reproduced by the addition of 8-bromo-cAMP, but not by the phorbol ester phorbol myristate acetate. In the presence of cyclohexamide, the effect of PTH on EGF binding was abolished, suggesting that new protein synthesis was required to increase the number of EGF receptors. Northern blots of total RNA, using a cDNA probe encoding the extracellular domain of the rat EGF receptor, revealed that PTH treatment resulted in a 2- to 3-fold increase in the level of EGF receptor mRNA. These data suggest that the proliferative effects of PTH on the osteoblast may be mediated indirectly by a PTH-induced increase in the number of EGF receptors.

The rat osteogenic sarcoma subclone UMR-106-01 is a cell type with osteoblast-like properties. This cell line has been shown to process specific receptors for insulin and insulin-like growth factor I (IGF-I), but not IGF-II. Insulin at physiological concentrations (1-5 ng/ml) in serum-free medium can maintain cell growth, as assessed by protein accumulation, thymidine uptake, and an increase in cell number. IGF-I is less potent than insulin, but, based on relative binding affinities for the insulin receptor, possibly acts via its own receptor. Insulin also enhances PTH-stimulated cAMP accumulation in these cells both by increasing cell number and an effect independent of cell number. Insulin may have a role in bone homeostasis.

We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.

Two receptors [estrogen receptor (ER)alpha and ERbeta] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ERalpha in the classical effects of estrogen activity, the physiological role of ERbeta is less well understood. A small-molecule ERbeta selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.

Thiazolidinedione analogs are new antidiabetic agents that attenuate peripheral insulin resistance in noninsulin-dependent diabetic patients; however, the effects of these agents on insulin secretion are not known. We determined the short-term and long-term effects of troglitazone (CS-045) on insulin secretion in a Syrian hamster clonal beta-cell line, HIT-T 15 cells. The direct effect of troglitazone (CS-045: 10(-6)-10(-4) M) on insulin secretion was examined in F-12 K incubation medium containing 7 mM glucose. CS-045 significantly stimulated insulin secretion within 10 min at the concentration of 10(-4) M and dose dependently stimulated insulin secretion within 60 min at the concentration of 10(-6)-10(-4) M. The addition of 10(-5) M CS-045 showed an immediate increase of cytoplasmic free Ca2+ concentrations ([Ca2+]i). Removal of extracellular Ca2+ by the addition of 1.5 mM EGTA completely abolished the 10(-4) M CS-045-induced insulin secretion for 10-min. Long-term incubation (24 h) with 10(-4) M CS-045 significantly decreased beta-cell insulin content and inhibited insulin secretion. During a 5-day incubation, CS-045 showed a dose-dependent reduction of insulin secretion measured during the final 24 h. Long-term incubation with CS-045 over 3 days inhibited the beta-cell proliferation rate, assessed with [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. CS-045 dose dependently increased the amount of DNA fragmentation measured by ELISA. The addition of nifedipine failed to attenuate the reduction of beta-cell proliferation rate and insulin secretion by CS-045, nifedipine antagonized an increase in the amount of DNA fragmentation caused by 10(-4) M CS-045. The present studies provide evidence that CS-045 inhibits beta-cell function following an acute stimulation of insulin secretion in HIT-T 15 cells. The immediate stimulation of insulin secretion by CS-045 may be mediated by an increase in Ca2+ influx from extracellular space. The induction of apoptosis may partially involves the reduction of beta-cell number by CS-045.

Recent evidence suggests that IL-1beta-mediated glucotoxicity plays a critical role in type 2 diabetes mellitus. Although previous work has shown that inhibiting IL-1beta can lead to improvements in glucose control and beta-cell function, we hypothesized that more efficient targeting of IL-1beta with a novel monoclonal antibody, XOMA 052, would reveal an effect on additional parameters affecting metabolic disease. In the diet-induced obesity model, XOMA 052 was administered to mice fed either normal or high-fat diet (HFD) for up to 19 wk. XOMA 052 was administered as a prophylactic treatment or as a therapy. Mice were analyzed for glucose tolerance, insulin tolerance, insulin secretion, and lipid profile. In addition, the pancreata were analyzed for beta-cell apoptosis, proliferation, and beta-cell mass. Mice on HFD exhibited elevated glucose and glycated hemoglobin levels, impaired glucose tolerance and insulin secretion, and elevated lipid profile, which were prevented by XOMA 052. XOMA 052 also reduced beta-cell apoptosis and increased beta-cell proliferation. XOMA 052 maintained the HFD-induced compensatory increase in beta-cell mass, while also preventing the loss in beta-cell mass seen with extended HFD feeding. Analysis of fasting insulin and glucose levels suggests that XOMA 052 prevented HFD-induced insulin resistance. These studies provide new evidence that targeting IL-1beta in vivo could improve insulin sensitivity and lead to beta-cell sparing. This is in addition to previously reported benefits on glycemic control. Taken together, the data presented suggest that XOMA 052 could be effective for treating many aspects of type 2 diabetes mellitus.

The UMR 106-06 rat osteosarcoma osteoblast-like cell line possesses calcitonin (CT) receptors in addition to expressing PTH receptors and a highly osteoblast-like phenotype, and may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts. Therefore, we examined the effects of CT and PTH on second messenger generation and osteoblastic function in these cells. In UMR-106-06 cells, 10-1000 nM CT produced a dose-dependent stimulation of intracellular free calcium concentration ([Ca2+]i), which reached a plateau between 2-3 min. This stimulatory effect was abolished in the absence of extracellular Ca2+ ([Ca2+]o) and was mimicked by forskolin and (Bu)2cAMP. One hundred nanomolar CT also produced a slight but significant increase in inositol triphosphate production (13%, P less than 0.05) but did not produce a rapid, transient increase in [Ca2+]i. In contrast, PTH produced a rapid, transient increase in [Ca2+]i, which reached a maximum within 30 sec. This stimulatory effect of PTH on [Ca2+]i signal was dose-dependent and accompanied by a parallel stimulation of inositol triphosphate production. PTH, forskolin, and (Bu)2cAMP all produced a marked dose-related suppression of both DNA and collagen synthesis, which paralleled their stimulatory effects on intracellular cAMP levels. In marked contrast, CT only minimally reduced DNA and collagen synthesis despite producing comparable increases in intracellular cAMP. One hundred nanomolar CT also stimulated alkaline phosphatase specific activity by 33% (P less than 0.05). Thus, CT stimulates cAMP, [Ca2+]i, and inositol phosphate second messengers in UMR 106-06 cells. However, in contrast to other agents which elevate intracellular cAMP levels, CT does not suppress DNA synthesis. These results suggest that the linkage of CT receptor second messengers to effects on cell function differ from those of PTH and/or that CT may produce additional second messenger(s) which antagonize the antiproliferative effect of increased cAMP levels in UMR-106-06 cells.

It has been well established that the spiroindoline sulfonamide MK-0677 stimulates GH secretion from the pituitary both in vitro and in vivo. MK-0677 has also been shown to increase serum insulin-like growth factor I (IGF-I) and cortisol levels in vivo; these increases are assumed to be driven by the increased serum GH and ACTH levels, respectively. However, such increases could also be due to a direct stimulatory action of MK-0677 at the level of the liver and adrenal cortex. To address this possibility, we investigated whether MK-0677 increased IGF-I and cortisol levels in hypophysectomized dogs. Baseline GH, IGF-I, and cortisol responses to MK-0677 (1 mg/kg, orally) were initially determined. Hypophysectomy (hypox; n = 7) or sham surgery (sham; n = 5) was then carried out. Six days postsurgery, the GH and cortisol responses to MK-0677 were reevaluated in each dog. In addition, each dog was treated with porcine GH (PST; 0.1 IU/kg, s.c.) to confirm the responsiveness of the GH-IGF-I axis. The mean peak GH increases in response to MK-0677 in the presham dogs (83.7 +/- 19.2 ng/ml), post-sham dogs (108 +/- 26.2 ng/ml), and pre-hypox dogs (121.2 +/- 13.6 ng/ml) were not significantly different. Mean peak GH levels were unchanged after MK-0677 administration in the hypox dogs (2.3 +/- 0.7 ng/ml). Before surgery, serum IGF-I levels increased to 243 +/- 27 and 224 +/- 47 ng/ml in the sham and hypox groups, respectively, after MK-0677 administration. Surgery was associated with a marked (> or =50%) decrease in serum IGF-I levels. MK-0677 administration increased IGF-I levels in the sham dogs from 78 +/- 14 to 187 +/- 31 ng/ml, whereas IGF-I levels remained unchanged (17.7 +/- 2.4 ng/ml) in the-hypox dogs. PST treatment increased IGF-I levels in the sham dogs from 162 +/- 30 to 325 +/- 32 ng/ml. In the hypox dogs PST treatment restored IGF-I to physiological levels (from 17.7 +/- 2.4 to 199 +/- 41 ng/ml). Cortisol was increased after MK-0677 administration 3.7-fold in the pre-sham, 3.6-fold in the post-sham, and 3.6-fold in the pre-hypox dogs, but no increase was seen in the post-hypox dogs. ACTH GEL administration (2.2 U/kg, i.m.) to hypox dogs returned cortisol to normal physiological levels, demonstrating the functional integrity of the adrenal cortex. This study demonstrates that the GH secretagogue MK-0677 does not directly stimulate an increase in serum IGF-I or cortisol levels, but depends upon the presence of an intact pituitary.

MK-0677, a spiroindoline sulfonamide, is a novel, orally active GH secretagogue. The effects of MK-0677 on serum GH and other hormones after oral and iv single dose administrations in beagles were evaluated. After oral administration in a balanced eight-dog crossover study, treatment with MK-0677 significantly increased peak GH concentrations, with a 5.3-fold increase (mean +/- SEM, 10.5 +/- 1.9 ng/ml) at the 0.25 mg/kg dose, a 9.0-fold increase (18.0 +/- 3.3 ng/ml) at the 0.50 mg/kg dose, and a 15.8-fold increase (31.6 +/- 5.8 ng/ml) at the 1.0 mg/kg dose. Total GH release, expressed as the area under the curve, showed similar significant increases over the effect of the water placebo. A single oral 1 mg/kg dose in three dogs induced a mean GH peak of 27.6 +/- 1.5 ng/ml at 120 min, and GH levels remained elevated up to 360 min after treatment. Insulin-like growth factor I (IGF-I) levels were significantly increased by 30% at 480 min after treatment. Cortisol levels were increased 2.4-fold over pretreatment levels. After i.v. administration, compared to the saline control group which had a mean (+/- SEM) serum GH peak of 3.8 +/- 0.7 ng/ml, MK-0677 at 0.25 mg/kg significantly increased (P < 0.05) peak GH concentrations 20.4-fold (77.4 +/- 13.7 ng/ml). Total GH release, expressed as the area under the curve, showed a similar increase. The mean peak GH level was recorded 10 min after treatment, with GH levels elevated up to 180 min after treatment. IGF-I levels were significantly elevated by 25% 360 min after the administration of MK-0677. Cortisol levels were increased 2.3-fold over pretreatment levels. Insulin and glucose levels were higher, LH and PRL levels were unaltered, and T4 levels were marginally lower; the levels of each of these hormones remained within the normal ranges for dogs throughout the experiment. In summary, MK-0677 is a potent GH secretagogue that induces an immediate, large, long lasting increase in GH levels when administered orally or i.v. In contrast to GH-releasing peptide-6 and benzolactam secretagogues, GH levels were elevated up to 360 min after treatment, and this was associated with a significant increase in IGF-I levels. Cortisol levels were increased; however, the increases were modest compared to those in GH.

Rats (200-275 g) were rendered pseudopregnant on the morning of estrus (day 1) by cervical stimulation. On day 5 of pseudopregnancy (PS), the rats (8-12/group) were subjected to either hysterectomy (HX) or unilateral or bilateral decidual cell reactions (uni- or bi-DCR). Control pseudopregnant (C-PS) rats were sham operated on day 5. On days 8 and 12, the rats were killed by decapitation. Blood was collected, and serum was saved for RIA of estradiol (E2), progesterone (P), androgen (A), and gonadotropins. Corpoa lutea (CL) and the nonluteal ovarian compartment (NLO) were incubated separately in vitro for 2 h in Krebs-Ringer bicarbonate buffer, and steroid concentrations were determined in Cl and NLS (before and after incubation) and in incubation media. On day 8 of PS, P and A levels in the serum in bi-DCR rats were higher than those in C-PS but not in uni-DCR and HX-PSP rats; however, on day 12, serum P and A levels in bi-DCR rats were higher than the levels of other PS groups, with the exception of C-PS rats whose serum A levels were the same as those in bi-DCR rats. No differences were observed in serum E2 levels throughout the experimental period. On days 8 and 12 the luteal P concentration of bi-DCR rats was higher than that of any other PS group, i.e. uni-DCR, HX-PS, and C-PS rats. The higher concentrations of luteal P in bi-DCR rats were associated with neither concomitant increases of luteal A and E2 nor higher serum levels of FSH, LH, and PRL, and they are therefore attributed to the large amount of decidual tissue (DT) in bi-DCR rats. In bi-DCR uni-DCR, and HX-PS rats, no differences were observed in the luteal concentration of A on day 8 or 12; however, E2 levels in CL of all PS groups were higher on day 12 than on day 8. The reasons for that difference is unclear, but it may be related to the onset of LH dependency of these Cl and, therefore, causally related to luteal maintenance. Collectively, these results indicate the possibility that DT selectively stimulates luteal P production in vivo without altering luteal A and E2, and that this effect of DT may be quantitatively related to the amount of DT. In vitro, CL of bi-DCR rats produced P, A, and E2 in quantities similar to other PS groups, with the exception of C-PS rats on day 12 whose CL were regressed and synthesized less P and more E2 than bi-DCR rats; therefore, a DCR effect on steroidogenesis in vitro was not observed. On day 12 of PS, a significant increase in vitro A and E2 production by NLO was observed, indicating that antral follicles may become highly competent to produce A and E2 during the latter portion of PS.

The mechanism(s) of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on rat (r) GH release was studied in primary rat pituitary cell cultures. TPA stimulated rGH release (3.2- to 4.1-fold above control value) and rTSH and rLH release (1.4- and 1.7-fold above control values, respectively), but not rPRL release. The ED50 of TPA on rGH secretion was 1.3 X 10(-9) M compared to 4.5 X 10(-11) M for human pancreatic GH-releasing factor [hpGRF-(1-44)]. If maximally effective doses of TPA or hpGRF-(1-44) were added to the cells, the magnitudes of the increase in rGH release were quite similar for both agents when the incubation period was less than 12 h. When (Bu)2cAMP was added simultaneously with various doses of TPA, (Bu)2cAMP increased rGH release beyond the maximal effect of TPA. There was an additive effect when hpGRF-(1-44) and TPA were used to stimulate rGH release. These results indicate that TPA enhances rGH release through a different pathway than hpGRF-(1-44). TPA failed to increase the formation of intra- and extracellular cAMP, whereas hpGRF-(1-44) increased both, suggesting that TPA stimulates rGH release through an cAMP-independent pathway(s). Protein kinase C has been postulated to be a receptor for TPA in human platelets. When phospholipase C, which activates protein kinase C via the formation of diacylglycerol, was added to the cells, rGH release was stimulated in a dose-dependent manner. This effect was not blocked by indomethacin. These results may suggest that activation of protein kinase C leads to rGH release. The observations are consistent with the hypothesis that TPA activates protein kinase C and causes the release of rGH in normal pituitary cells in culture. These findings indicate that the mechanism(s) of action of TPA on rGH release is different from that of hpGRF-(1-44).

The present studies were designed to determine the effects of ethane 1,1 diphosphoric acid (EHDP(TM)), an analog of pyrophosphate, on the hypocalcemic and hypophosphatemic actions of salmon calcitonin (SCT). Rats were pretreated for 3 or 4 days with daily subcutaneous (sc) injections of EHDP (40 mg/kg body wt). 45Ca was administered (intraperitoneally) 10 days prior to EHDP treatment and 32P (intravenously) the day following the last injection of the drug. SCT (0.4 MRC mU/g body wt) was injected sc 1 hr after 32P administration. Preliminary tests with this drug had confirmed that the dosage used at least partially inhibited the 45Ca removal from bone stimulated by low calcium diet or parathyroid hormone administration, and at least partially reduced the uptake of both 45Ca and 32P by bone, during the first 5 hr after intravenous administration of isotopes. Following SCT injection plasma calcium concentrations fell only 5% in EHDP treated rats compared to 25% in rats not treated with EHDP. Plasma phosphate concentrations and 32P plasma values, however, fell markedly and similarly in rats given SCT regardless of whether or not they had been pretreated with EHDP. These studies demonstrate that the hypophosphatemic effect of SCT was produced independently of the hypocalcemic action of the hormone and the hypophosphatemia did not result solely from a reduction of phosphate release from bone but rather was due to an increased exit of phosphate out of the plasma.

Concern that some chemicals in our environment may affect human health by disrupting normal endocrine function has prompted research on interactions of environmental contaminants with steroid hormone receptors. We compared the activity of 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor, at estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). Human hepatoma cells (HepG2) were transiently transfected with either human or rat ERalpha or ERbeta plus an estrogen-responsive, complement 3-luciferase construct containing a complement 3 gene promoter sequence linked to a luciferase reporter gene. After transfection, cells were treated with various concentrations of HPTE in the presence (for detecting antagonism) or absence (for detecting agonism) of 17beta-estradiol. HPTE was a potent ERalpha agonist in HepG2 cells, with EC50 values of approximately 5 x 10(-8) and 10(-8) M for human and rat ERalpha, respectively. In contrast, HPTE had minimal agonist activity with either human or rat ERbeta and almost completely abolished 17beta-estradiol-induced ERbeta-mediated activity. Moreover, HPTE behaved as an ERalpha agonist and an ERbeta antagonist with other estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa cells. This study demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.

1,10-Phenanthroline, a metal ion chelator, inhibits the binding of previously activated (25 C for 30 min) rat hepatic [3H]triamcinolone acetonide (3H-labeled 9-fluoro-11 beta, 21-dihydroxy-16 alpha, 17-1-[1-metylethylidenebis(oxy)]pregna-1,4-diene-3,20-dione ([3H]TA)-receptor complexes to DNA-cellulose. The observed inhibition increases as the temperature of the preincubation with chelator is increased from 0 to 25 C. Fifty percent of the maximal inhibition (greater than 90%) detected at 25 C is achieved with 1 mM 1,10-phenanthroline. The observed inhibition is not the consequence of DNA degradation by 1,10-phenanthroline-Cu2+ complexes, since preincubation of activated cytosol with neocuproine (2,9-dimethyl-1,10-phenanthroline), a potent Cu2+ chelator, fails to block the subsequent inhibition of DNA-cellulose binding by 1,10-phenanthroline. The failure of other chelators which complex siilar metal ions (alpha, alpha'-dipyridyl,8-hydroxyquinoline, 2,2',2"-tripyridine, EDTA, EGTA, and Na azide) to inhibit DNA-cellulose binding suggests that the effectiveness of 1,10-phenanthroline does not result from removal of a required free metal ion(s) but, rather, from a specific interaction with a metal ion(s) which may be located within the activated receptor protein. The observed inhibition is dependent on the metal chelating properties of 1,10-phenanthroline, since preincubation with several divalent metal cations (Zn2+, Co2+, and Ni2+) which are known to be chelated by this compound block its subsequent inhibitory effect. Ferroin (1,10-phenanthroline-ferrous sulfate complex) and 1,7-phenanthroline (nonchelating isomer) also fail to inhibit DNA-cellulose binding. The inhibition mediated by 1,10-phenanthroline persists after gel filtration, suggesting that 1,10-phenanthroline associated with a macromolecule is the effective form of the inhibitor, rather than free 1,10-phenanthroline. Finally, 1,10-phenanthroline appears to interact directly with activated [3H]TA-receptor complexes, since it alters their net charge and results in their elution from DEAE-cellulose at a salt concentration characteristic of unactivated complexes. Collectively, the data suggest that the activated [3H]TA-receptor complex is a metalloprotein and that the metal ion(s) may be associated directly with the DNA-binding site or may regulate this site indirectly through an allostreic mechanism.

The transmembrane signaling events of GH were investigated in the liver, a major target organ of GH action. Recombinant human GH when added to freshly isolated rat hepatocytes rapidly stimulated the production of sn-1,2-diacylglycerol (DAG). The generation of DAG was biphasic with the first transient peak observed at 2 min and the second peak at 15 min (1.2-fold and 1.4-fold over control, respectively). Levels of DAG continued to be elevated above those in control cells at 30 min. The response was dose-dependent with an EC50 of 0.15 nM. Both bovine GH and rat GH, which bind to the rat GH receptor but not to the PRL receptor, also stimulated DAG production. Similarly, human PRL, which binds to the PRL but not GH receptor, stimulated DAG formation to a comparable extent. These results suggest that production of DAG may be an early signaling event mediated by hormone stimulation of both the GH and PRL receptors.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was examined for a possible stimulative effect on osteoblastic MC3T3-E1 cells. During the early period of culture, 1,25-(OH)2D3 had a stimulative effect. During the growth phase, however, the steroid had little effect on either the protein or DNA content of the cultures. 1,25-(OH)2D3 increased bone-liver-kidney-type alkaline phosphatase activity in a dose-related manner up to a concentration of 5 pg/ml; the increase was 2.2-fold over the control value. Studies on the effect of actinomycin D or cycloheximide treatment indicated that the vitamin may enhance de novo synthesis of ALP. The steroid also stimulated type I collagen production dose dependently via an increase in collagen synthesis rather than by inhibition of collagen degradation. MC3T3-E1 cells have a specific receptor for 1,25-(OH)2D3 which has a dissociation constant of 4.17 X 10(-11) M and a sedimentation coefficient of 3.67S. The receptor concentration varied with the period of culture, being higher during the growth phase and lower at confluence, but its affinity did not change. The results indicate that 1,25-(OH)2D3 has a direct specific anabolic effect on osteoblastic cells in vitro during the growth phase and that this effect is related to receptor concentration.