Showing papers in "Endocrinology in 1987"

Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat.

Abstract: The insulin-like growth factors (IGFs) have both metabolic and growth-promoting activities in many cell and tissue types. Although the IGFs are present in serum, they are also thought to have important autocrine and paracrine functions. Using complementary DNA (cDNA) probes for rat IGF-I and mouse IGF-II, we have investigated the tissue distribution of messenger RNAs (mRNAs) for these growth factors in adult rats. IGF-I cDNA hybridized with three groups of transcripts, 7.0, 1.8 and 0.7-1.1 kilobases, which were detectable in all tissues examined, with liver demonstrating the highest level of expression. IGF-II cDNA also hybridized to a number of mRNAs, the most abundant of which was 4.0 kilobases. Of the tissues examined, IGF-II expression was highest in the brain, barely detectable in the liver, and undetectable under the conditions used, in lung, ovary, testes, and mammary gland. These studies support the notion of paracrine or autocrine function for IGF-I and demonstrate tissue-specific IGF-II expression in the adult rat.

Interleukin-1 stimulates ACTH release by an indirect action which requires endogenous corticotropin releasing factor.

Abstract: The present study was performed in order to clarify the mechanism by which interleukin-1 (IL-1) activates the hypothalamic-pituitary-adrenal (H-P-A) axis. The iv administration of IL-1 into freely moving, conscious rats significantly elevated the plasma levels of ACTH. This ACTH response to IL-1 was, however, completely abolished by preinjection of 0.5 ml rabbit antiserum generated against rat CRF, but not by normal rabbit serum (NRS). The IL-1-induced ACTH release did not seem to be caused by a general stress effect of IL-1 because plasma PRL levels, another indicator of a stress response, were not altered by the injection of IL-1. These results suggest that IL-1 acts centrally in the brain to stimulate the secretion of CRF, thereby eliciting ACTH release, and that a direct action of IL-1 on the pituitary gland is unlikely.

Localization and Characterization of Insulin Receptors in Rat Brain and Pituitary Gland Using in Vitro Autoradiography and Computerized Densitometry

Abstract: In order to identify likely sites of action in insulin in rat brain we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize, and quantify its receptors in coronal and sagittal sections A discrete and characteristic distribution of insulin receptor binding was demonstrated, with specific binding representing 92% of total binding Displacement and specificity competition curves in olfactory bulb are typical for authentic insulin receptors, and computer analysis indicates a single class of binding site with a dissociation constant (Kd) 048 nM for choroid plexus and 044 nM for olfactory bulb external plexiform layer Insulin receptor density is maximum in the choroid plexus, and high in the external plexiform layer of olfactory bulb Structures of the limbic system and hypothalamus reveal moderate to high insulin receptor density, particularly the lateral septum, amygdala, subiculum, hippocampal CA1 region, mammillary body, and arcuate nucleus Moderate ins

Facilitation of immunoreactive corticotropin-releasing factor secretion into the hypophysial-portal circulation after activation of catecholaminergic pathways or central norepinephrine injection.

Abstract: The functional role of central catecholamines in regulation of ACTH secretion remains controversial. In the present report, the nature of catecholaminergic influences was directly assessed by measurement of hypophysial-portal plasma immunoreactive CRF (irCRF) levels after activation of endogenous aminergic pathways by electrical stimulation or administration of norepineprhine (NE). Electrical stimulation of the ventral noradrenergic ascending bundle, a fiber system primarily carrying catecholaminergic fibers arising from brainstem regions, resulted in a 2.9-fold elevation of portal irCRF levels. Pretreatment with the αl-adrenergic receptor antagonist coryanthine, but not the β-adrenergic antagonist propranolol, blocked the facilitatory effect of electrical stimulation and reduced prestimulation irCRF levels by 34.7 ± 4.2% (P < 0.05). Intracerebroventricular administration of 0.1-5.0 nmol NE resulted in a dose-dependent facilitation of portal plasma irCRF levels which could be blocked by pretreatment with ...

Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro.

Abstract: Isolated rat calvaria cells plated at low density in medium supplemented with ascorbic acid and organic phosphate form discrete three-dimensional mineralized nodules having the characteristics of bone. We have studied the effects of glucocorticoids on the formation of bone nodules by these cell populations. Cells isolated from 21-day-old fetal rat calvaria were maintained in vitro for up to 27 days. Dexamethasone (Dex) induced a dose-related increase in the number of nodules formed, with a peak at 10 nM and a half-maximal response at about 1 nM. Dex (10 nM) also significantly increased the size of bone nodules formed (P less than 0.002). High concentrations of Dex (1 microM) did not increase nodule number. In cells in primary culture maintained in medium containing 10 nM Dex, the increase in nodule number was 50-100% over the control value. The effect of Dex was much greater in first subculture cells, where the number of nodules was 600-800% higher than the control value. Dishes collected and quantitated from 12-27 days showed that nodule formation ceased between 15 and 18 days in cultures without Dex, whereas in the presence of Dex the number of nodules increased up to 27 days. Addition of 10 nM Dex only during specific periods resulted in significantly more nodules than in control cultures, but significantly fewer nodules than in cultures constantly exposed to Dex. Cell population doubling times during log phase growth were unaltered, but a significant increase in saturation density (P less than 0.001) was observed with 10 nM Dex. Hydrocortisone also caused an increase in the number of nodules formed, with a maximal effect of 50 nM and a half-maximal response at 8 nM. The results indicate that physiological levels of glucocorticoids stimulate bone nodule formation in long term cell culture by increasing the number of cells forming bone nodules and that maximization of the stimulatory effect of glucocorticoids on bone formation may require constant exposure to low levels of the hormone.

Inhibitory effects of interleukin 1 on insulin secretion, insulin biosynthesis, and oxidative metabolism of isolated rat pancreatic islets.

Abstract: Recent observations suggest a role for interleukin 1 (IL-1), a macrophage-derived cytokine, in the autoimmune B cell destruction, which is observed in type 1 diabetes. In the present study we have investigated the effects of IL-1 and two other cytokines, namely tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) on the pancreatic B cell paying particular attention to insulin production and glucose metabolism. Rat pancreatic islets were isolated and kept in tissue culture for 5 days. The islets were subsequently transferred to media containing medium RPMI 1640 plus 0.5% human serum with or without additions of human recombinant preparations of either IL-1 (25 U/ml), TNF (1000 U/ml), or IFN-gamma (500 U/ml), and cultured for another 48 h. After the culture period the islets were subjected to light microscope examination and different functional tests in short-term incubations in the absence of cytokines. IL-1 was found to reduce insulin release in culture and totally inhibit glucose-stimulated insulin release in short-term incubations. Islet (pro)insulin biosynthesis, glucose oxidation, and oxygen uptake at 16.7 mM glucose were partially inhibited by IL-1. The DNA content of islets cultured with IL-1 was decreased and may partly explain these latter findings. However, inhibition of glucose oxidation could not be seen in islets exposed to IL-1 in short-term experiments only. By light microscopy there were marked signs of degeneration in IL-1 treated islets. TNF and IFN-gamma were essentially without effect on islet morphology or function. The results of this study indicate that IL-1 may be cytotoxic to islet B cells. The primary toxic action of IL-1 seems to involve factors other than an impaired islet glucose metabolism.

Corticotropin-Releasing Factor Decreases Plasma Luteinizing Hormone Levels in Female Rats by Inhibiting Gonadotropin-Releasing Hormone Release into Hypophysial-Portal Circulation

Abstract: To evaluate whether the hypothalamus is the site of action of CRF in inhibiting LH levels in female rats, we measured hypophysial-portal blood concentrations of immunoreactive GnRH (irGnRH) after the central injection of CRF. Ovine CRF (0.1, 1.0, 2.0, and 5.0 nmol) was injected intracerebroventricularly to intact rats on the afternoon of proestrus and in long term ovariectomized (OVX) rats in the presence or in absence of estradiol benzoate (OVX + EB). CRF injection decreased the amplitude of the proestrous irGnRH surge without affecting presurge levels. CRF (0.1 nmol) attenuated the afternoon irGnRH surge in OVX + EB rats; higher doses of CRF blocked this surge and decreased nonsurge irGnRH levels. No dose-related alterations of irGnRH levels were observed in OVX rats; only the highest dose of CRF was active. For comparison, plasma LH concentrations were measured after a single dose of CRF (2 nmol) in rats under the same experimental conditions. While CRF decreased LH concentrations in anesthetized proes...

Lipid peroxidation and free radical scavengers in thyroid dysfunction in the rat: a possible mechanism of injury to heart and skeletal muscle in hyperthyroidism.

Abstract: This study was designed to determine if peroxidation of biomembrane lipid and the protective system can be modified by the change in oxidative metabolism induced by thyroid dysfunction. The free radical scavengers (i.e. cuprozinc cytosolic and mangano mitochondrial superoxide dismutases, glutathione peroxidase, and catalase), mitochondrial oxidative marker enzymes (cytochrome c oxidase and fumarase), and lipid peroxide were measured in liver, heart, soleus (slow oxidative), and extensor digitorum longus (fast glycolytic) muscles. Rats were rendered hyper- or hypothyroid for 4 weeks and then killed. Superoxide dismutases were detected by specific RIAs: catalase by polarography, and lipid peroxide by fluorimetry. Hypothyroid rats failed to grow, while hyperthyroid rats had hypertrophied hearts but no growth failure. An increase in lipid peroxide was observed in the soleus and heart muscles of hyperthyroid rats. This was accompanied by an increase in mitochondrial superoxide dismutase and oxidative markers. ...

A Comparative Study of the Distributions of Renin and Angiotensinogen Messenger Ribonucleic Acids in Rat and Mouse Tissues

Abstract: Previous studies have reported the presence of renin mRNAs in several mouse tissues and angiotensinogen mRNAs in various rat tissues. Clarification as to whether renin and angiotensinogen mRNAs are coexpressed in the same tissues of the same animal species is important for understanding the biology of the tissue renin-angiotensin system. We employed mouse renin cDNA and rat angiotensinogen cDNA to compare tissue distributions of renin and angiotensinogen in RNAs of the rat and mouse. Both cDNA probes readily cross-hybridize with the corresponding mRNA of the other species. Our results demonstrate several patterns of distribution. 1) Renin and angiotensinogen mRNAs are readily detected in kidney and adrenals of both species. In brain and heart, angiotensinogen mRNAs are present in concentrations that far exceed renin mRNA levels in these organs in both species. 2) In mouse and rat livers, angiotensinogen, but not renin, mRNA is demonstrated. 3) In rat testis, only renin mRNA can be detected, whereas in mou...

Gonadotropins and Estradiol Stimulate Immunoreactive Insulin-Like Growth Factor-I Production by Porcine Granulosa Cells in Vitro*

Abstract: Previous studies have established the ovarian granulosa cell as a site of insulin-like growth factor-I (IGF-I) secretion and action, suggesting an autocrine function for this peptide in the ovary. To better understand how this putative autocrine system is regulated and its interface with the classic ovarian trophic hormones FSH, LH, and estradiol (E2), we have studied the effects of these hormones on the secretion of immunoreactive IGF-I (iIGF-I) by cultured porcine granulosa cells. Immature granulosa cells were cultured under serum-free conditions which were optimized to allow maximal iIGF-I production and hormonal responsivity. Measurements of iIGF-I were made after minimizing the influence of IGF-binding proteins by either acid gel filtration or reverse phase chromatography. Since the two preparative procedures gave roughly comparable results, the more expeditious reverse phase procedure was chosen for most samples. Cycloheximide virtually eliminated measurable iIGF-I in culture, suggesting that the peptide measured was newly synthesized, and degradation of IGF-I by cultured granulosa cells was negligible. Consequently, the medium levels provided an accurate indication of cellular secretion over the collection period. Under optimal culture conditions, iIGF-I was readily measurable and responsive to treatment with ovarian trophic hormones. The iIGF-I levels in several experiments with these hormones were as follows: FSH treatment, 1.58 +/- 0.21 times the control value (n = 5 experiments); E2 treatment, 1.26 +/- 0.12 times the control value (n = 5); E2 plus FSH, 3.12 X 0.31 times the control value (n = 8); LH, 1.33 +/- 0.12 times the control value (n = 3); LH plus FSH, 1.78 +/- 0.2 times the control value (n = 1). To assess the role of cAMP in the mediation of gonadotropin effects in this system, granulosa cells were treated with a phosphodiesterase inhibitor (methylisobutylxanthine), which resulted in iIGF-I levels 1.61 +/- 0.7 times the control level. In the presence of FSH, a further stimulatory effect was demonstrated (3.76 +/- 0.29 times control). In addition, the cAMP analog 8-bromo-cAMP dramatically increased iIGF-I levels (6.3 +/- 0.72 times control). These data provide the first demonstration that gonadal iIGF-I secretion can be stimulated by the principal hormones involved in trophic regulation of the ovary. As with other gonadotropin-dependent functions of granulosa cells, this effect appears to be mediated by cAMP and enhanced by E2. This interface between circulating hormones and autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones on a local level.

N-methyl-D,L-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male rhesus monkeys (Macaca mulatta).

Abstract: In higher primates, the protracted delay from infancy to puberty results from an interruption in hypothalamic GnRH release. To determine whether the quiescent hypothalamic GnRH neurons of the prepubertal macaque are capable of discharging the decapeptide in response to a generalized neural depolarization, an excitatory amino acid analog, N-methyl-D,L-aspartate (NMA), was administered systemically to orchidectomized rhesus monkeys between 13 and 20 months of age. GnRH secretion was estimated indirectly by monitoring changes in circulating LH concentrations after the responsivity of pituitary gonadotropes to GnRH had been greatly facilitated by the chronic intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every hour). The iv bolus administration of increasing doses of NMA (1.5, 4.8, and 15.0 mg/kg BW), 10-14 h after termination of the priming infusion of GnRH, elicited distinct discharges of LH, with magnitudes directly related to the amount of the excitant injected. Administration of a higher dose of NMA (48 mg/kg BW), however, failed to induce further LH release. The finding that pretreatment with a long-acting and potent GnRH receptor antagonist [( AcD2Nal1,4ClPhe2,DTrp3,DArg6,DAla10] GnRH-HOAc) abolished the LH-releasing activity of NMA provides compelling evidence for the view that the action of the neural excitant to induce gonadotropin release was exerted at a suprapituitary level. The additional observation that an N-methyl-D-aspartate receptor antagonist (D,L-2-amino-5-phosphono-valeric acid) blocked the NMA-induced release of GnRH suggests that the amino acid analog interacted with the N-methyl-D-aspartate receptor on neurons that synthesize and/or control the release of the hypothalamic hormone. Most interestingly, three sequential GnRH discharges, with a period and an amplitude apparently similar to those generated by the hypothalamus of the adult, were elicited from the brain of prepubertal monkeys by the sequential administration of three injections of NMA at hourly intervals. Taken together these findings demonstrate that the apparent dormancy of hypothalamic GnRH neurons, which is characteristic of prepubertal development in higher primates and underlies the protracted delay in the onset of puberty in these species, may be readily terminated by application of a generalized neural excitation. Plasma FSH, PRL, GH, and cortisol concentrations were also monitored during the course of some of these experiments, and release of each of these four hormones was observed after the iv injection of NMA (15 mg/kg BW).

A receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid.

Abstract: PRL interacts with areas of the central nervous system which reside behind the blood-brain barrier. While vascular PRL does not cross this barrier, it is readily accessible to the cerebrospinal fluid (CSF) from which it may gain access to the PRL-responsive areas of the brain. Studies were undertaken to characterize the mechanism responsible for the translocation of PRL from blood to CSF. Rats were given external jugular vein injections of [125-I]iodo-PRL in the presence or absence of an excess of unlabeled ovine PRL (oPRL), human GH, bovine GH, or porcine insulin. CSF and choroid plexus were removed 60 min later. CSF samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide slab gels and resultant autoradiographs were analyzed with quantitative microdensitometry. The data revealed that unlabeled lactogenic hormones, viz. oPRL and human GH, caused a statistically significant inhibition of [125I]iodo-PRL transport from blood to CSF. In contrast, nonlactogenic hormones, viz bovine GH and insulin, had no effect on [125I]iodo-PRL transport into the CSF. An identical pattern of competition was observed in the binding of hormone to the choroid plexus. Furthermore, vascular injections of [125I]iodo-PRL administered with a range of concentrations of unlabeled oPRL revealed a dose-response inhibition in the transport of [125I]iodo-PRL from blood to CSF. The study demonstrates that PRL enters the CSF by a specific, PRL receptor-mediated transport mechanism. The data is consistent with the hypothesis that the transport mechanism resides at the choroid plexus. The existence of this transport mechanism reflects the importance of the cerebroventricular system in PRL-brain interactions.

Hypophysial-portal plasma levels, median eminence content, and immunohistochemical staining of corticotropin-releasing factor, arginine vasopressin, and oxytocin after pharmacological adrenalectomy.

Abstract: Changes in immunostaining, median eminence content, and secretion into the hypophysial-portal circulation of immunoreactive CRF (irCRF), arginine vasopressin (irAVP), and oxytocin (irOT) were directly evaluated after pharmacological adrenalectomy (PHADX). Mean circulating levels of ACTH rose from 270 +/- 57 (+/- SE) to 1560 +/- 283 pg/ml after 72 h of treatment with metyrapone and aminoglutethimide. Initially, hypophysial-portal plasma irCRF levels decreased to 52.6% (12 h) and 21.7% (24 h) of control levels (230 +/- 41 pg/ml). Accompanying changes in the patterns of CRF immunostaining in the paraventricular nuclei (PVN) or in median eminence irCRF content at 24 h did not parallel alterations in portal plasma irCRF levels at this time. By 72 h posttreatment, portal irCRF levels were elevated 2.2-fold, while the number of detectable CRF-positive perikarya in the PVN increased 3.0-fold. The mean hypophysial-portal plasma irAVP concentration was unchanged from the control value (1312 +/- 287 pg/ml) at 12 h, but was only 34.9% of the control value at 24 h. Inverse changes in median eminence irAVP content were noted at these times, whereas the number of AVP-immunostained cells exhibited a tendency toward an increase at 24 h, in parallel with significantly increased content. By 72 h post-PHADX, portal irAVP, median eminence irAVP content, irAVP immunostaining intensity, and AVP-immunopositive cell number were elevated. Approximately 64% of CRF-positive perikarya in the parvocellular PVN costained for AVP at this time, whereas no colocalization was evident in untreated rats. These changes were prevented by corticosterone replacement. irOT staining intensity, irOT-positive cell number, median eminence irOT content, and portal plasma irOT concentration remained stable at all times examined. We conclude that: removal of adrenal steroids by PHADX results in a sequence of changes in CRF and AVP within the PVN (as determined by immunocytochemistry) and the median eminence (as determined by peptide content) similar to those observed after surgical adrenalectomy; after steroid removal the secretion of both irCRF and irAVP changes in a biphasic manner characterized by reduced secretion at 24 h and greatly enhanced secretion at 72 h; neither immunostaining nor median eminence content alone proved to be a reliable index or secretory activity during the initial phases of steroid blockade; and the hypophysiotropic OT system of normal male rats appears to be insensitive to adrenal steroid influences.

Identification, characterization, and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor-I.

Abstract: A complementary DNA (cDNA) encoding rat insulin-like growth factor 1 (IGF-I) has been isolated from a kidney cDNA library. By analogy with the sequence of human and mouse preproIGF-IA this cDNA encodes rat preproIGF-I from amino acid minus 3 to amino acid 105. The predicted protein sequence shows 96% and 99% homology with the human and mouse preproIGF-I, respectively. Under stringent conditions the rat IGF-I cDNA hybridizes with at least three mRNA, messenger RNA species which have apparent sizes of 7, 1.8, and 0.7-1.1 kilobases. All three IGF-I transcripts are detectable in each of the normal rat tissues examined and the relative order of abundance in tissues from intact adult male rats is liver greater than lung greater than kidney greater than thymus greater than spleen greater than heart greater than skeletal muscle (quadriceps femoris) greater than testes greater than brain. The GH dependence of the IGF-I mRNAs was demonstrated by the administration of a single ip injection of human GH (hGH) to hypophysectomized (hypox) rats which resulted in an increase in all three transcripts in each of the tissues examined. The increase in IGF-1 mRNAs was most marked in skeletal and cardiac muscle (9.7- and 9.5-fold compared to hypox controls, respectively) and least marked in the brain. In the liver only a 4-fold increase in IGF-I expression was observed, possibly because of the relatively high level of IGF-I expression in the tissue in the hypox control rats. Each of the IGF-I messenger RNAs appeared to increase in parallel and the time course of IGF-I induction was similar in each tissue with maximal levels of IGF-I transcripts present 6 to 12 h after GH administration. A dose-dependent increase in IGF-I mRNAs was observed in most tissues of hypox rats treated for 10 days with hGH. Significant correlations between growth, as determined by body weight gain and IGF-I expression were observed in liver (r = 0.97), kidney (r = 0.90), quadriceps femoris (r = 0.95), diaphragm (r = 0.92), and thymus (r = 1.0). The IGF-I mRNA levels in tissues from rats that had been treated with the highest dose of hGH (90 microgram/rat X day) were similar to those observed in normal intact rats. This study confirms the highly conserved nature of the IGF-I precursor and provides clear evidence for the GH dependence of IGF-I gene expression in multiple tissues of the rat.

Tumor Necrosis Factor-α (Cachectin) Stimulates Bone Resorption in Mouse Calvaria via a Prostaglandin-Mediated Mechanism

Abstract: Recombinant human (h) and murine (m) tumor necrosis factor (TNF)-alpha stimulated bone resorption and the production of prostaglandin (PG) E2 in neonatal mouse calvaria in organ culture. In experiments of 72-h duration, the effect on bone resorption was of large magnitude (an average increase in medium calcium of 3.3 mg/dl above control values in 11 separate experiments) and occurred over a concentration range of 0.1-20 ng/ml mTNF and 0.5-50 ng/ml hTNF. Accompanying the TNF-enhanced release of bone calcium there was enhanced accumulation of PGE2 in the culture medium. The increases in medium calcium and PGE2 were both inhibited completely by nontoxic concentrations of 4 different PG cyclooxygenase inhibitors (indomethacin, piroxicam, ibuprofen, and acetylsalicylic acid) but not by the noncyclooxygenase inhibitor salicylic acid. The magnitude of the PGE2 response, but not the calcium release, was less for bones treated with TNF than for those treated with equipotent doses of epidermal growth factor or human transforming growth factors-alpha or -beta, suggesting that the local site of production of PGE2 in bone may be different for TNF than for the other factors. Repeated sc injections of hTNF to intact mice for a 48-h period produced a statistically significant elevation of the plasma calcium concentration. Because TNF is produced by cells of the monocyte/macrophage lineage in response to invasive stimuli such as the presence of tumor, our findings indicate that a host factor produced in response to malignant cells can cause enhanced bone resorption. Thus, the concept of the humoral hypercalcemias of malignancy must be expanded to include mediators not produced by the tumor cells themselves.

Further evidence for a central stimulatory action of catecholamines on adrenocorticotropin release in the rat.

Abstract: Catecholamines may stimulate ACTH secretion during stress. To investigate the nature and site of such an action, plasma ACTH was measured in four groups of unanesthetized adult female rats with an indwelling carotid cannula. Sequential 300-microliter blood samples were taken 60 min, 30 min, and immediately before an intracerebroventricular (icv) infusion of 2.5 microliter adrenaline or noradrenaline and 5, 15, 45, 60, and 120 min after the infusion. The four groups were: 1) intact rats; 2) rats infused 7 days after undergoing a discrete bilateral lesion of the ventral noradrenergic ascending bundle caused by 6-hydroxydopamine, which depleted their hypothalamic adrenaline and noradrenaline levels by 90% and 80%, respectively; 3) rats infused 30 min after pretreatment via the icv route with either prazosin or propranolol; and 4) rats infused 16 and 2 h after two successive intracarotid injections of an anti-rCRH-41 serum. In another group, the effects of icv catecholamine administration were compared with those of an intracerebral (ic) microinfusion close to a single paraventricular nucleus (PVN). Finally, in two additional groups blood was sampled at the above-mentioned times before and after a 2-min ether inhalation by intact rats or prazosin- and/or propranolol-pretreated rats. In the intact rats (group 1), a stress-like stimulatory dose response was noted after both adrenaline and noradrenaline infusions, with a half-maximal effect at concentrations of about 0.6 nmol and a maximal effect at 2.7 nmol or more. At maximally effective doses, adrenaline was significantly more active than noradrenaline. In the rats with ventral noradrenergic ascending bundle lesions (group 2), 2.7 nM adrenaline or noradrenaline stimulated ACTH release as in the controls without lesions. In group 3, prazosin blocked the ACTH responses to both adrenaline and noradrenaline, whereas propranolol only blocked the response to adrenaline. In group 4, i.e. rats pretreated with an anti-rCRH-41 serum, the amplitude of the ACTH surge after icv adrenaline or noradrenaline infusion was halved. A unilateral ic catecholamine microinfusion next to the PVN (half the icv dose given in group 1) led to a rapid ACTH release that peaked at half the response measured in the icv infused rats. Ether stress-induced ACTH release was decreased by 50-60% after icv pretreatment with 1 or 10 micrograms prazosin, 1 or 6.5 micrograms propranolol, or a combined dose comprising 1 microgram of both. The following conclusions were reached.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Transforming Growth Factor-β on Osteoblastic Osteosarcoma Cells*

Abstract: Transforming growth factor-beta (TGF beta), a polypeptide that controls growth and differentiation in many cell types and has recently been found in abundant amounts in bone, was examined for its effects on cells with the osteoblast phenotype using the clonal osteoblastic osteosarcoma cell line ROS 17/2.8. TGF beta increased alkaline phosphatase (AP) activity and the rate of collagen synthesis per cell. Cell proliferation was inhibited, and the morphological appearance of the cells was markedly changed. All effects were observed at concentrations as low as 0.1 ng/ml TGF beta. Increases in AP activity were detectable after 24 h and increased progressively with time. TGF beta increased AP activity under serum-free conditions and during thymidine-induced inhibition of DNA synthesis. The increase in AP activity mediated by TGF beta could be completely inhibited with actinomycin D and cycloheximide. 1,25-Dihydroxyvitamin D3 at 10(-7) M slightly increased AP activity in ROS 17/2.8 cells, but strongly inhibited AP activity when the cells were pretreated with TGF beta. The data suggest that TGF beta stimulates expression of the osteoblastic phenotype in ROS 17/2.8 cells and that TGF beta may be an important regulator of local bone remodeling.

The type II insulin-like growth factor (IGF) receptor has low affinity for IGF-I analogs: pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor.

Abstract: We have characterized binding and several actions of the somatomedins [insulin-like growth factors I and II (IGF-I and IGF-II)] on L6 myoblasts. Both IGF-I and IGF-II are potent stimulators of amino acid uptake, cell proliferation, and differentiation; they also suppress protein degradation in these cells. In all measurements, the relative potencies are IGFI > IGF-II > insulin. Two recombinant DNA-produced analogs of IGF-I, (Thr69)IGF-I and (N-Met)IGF-I, were as active as native IGF-I in all four assays. However, when 125I-labeled hormones were used for studies of binding to IGF receptors, there was a striking difference between the native and recombinant IGF-I molecules. Both were bound significantly by the type I receptor (a 350K molecule that is dissociated upon sulfhydryl reduction), but the recombinant analogs exhibited little cross-reactivity with the type II receptor (a 220K molecule that is not dissociated by reduction). Thus, the equal activity of native IGF-I and its recombinant DNA-produced ana...

Hormonal control of pubertal spermatogenesis.

Abstract: The spermatogenic process of normal rats at 20, 32, and 44 days of age was characterized. Variations in numbers of degenerating and abnormal cells were noted during the cycle in most age groups, indicating a stage-related vulnerability of these cells. The most advanced cell types that were seen at a particular age were frequently abnormal or degenerating. When the numbers of viable cells available to degenerate were considered, the degeneration rate in normal pubertal animals was about 15, 10, and 2 times greater in 20-, 32-, and 44-day-old animals, respectively, than in 75-day-old animals. In 32-day-old rats, neither hypophysectomy nor hypophysectomy and subsequent hormone supplementation resulted in an alteration in the qualitative pattern of germ cell degeneration during the spermatogenic cycle compared with that in the normal animal; however, the treatments did alter the quantitative response of cellular degeneration. Three days posthypophysectomy there was a marked increase in the numbers of total degenerating germ cells. FSH (60 micrograms) given twice daily (as were all hormones) reduced the numbers of degenerating cells significantly, as did LH (13 micrograms). Low dose LH (0.3 micrograms), representing the approximate contaminating dose of LH in the 60-micrograms FSH preparation, and low dose FSH (30 micrograms) did not elicit a response significantly different from that to hypophysectomy alone. LH (13 micrograms) plus FSH (60 micrograms) reduced the levels of degenerating cells such that there was no significant difference from levels in intact 32-day-old rats. The data indicated, for the cell types studied, a lack of specificity of various hormones or hormone combinations in the survival of specific germ cell types. It emphasizes the importance of FSH in pubertal spermatogenesis as well as the synergistic actions of LH and FSH.

Intracellular Localization of the Glucocorticoid Receptor: Evidence for Cytoplasmic and Nuclear Localization*

Abstract: Using a monospecific, monoclonal antibody against the glucocorticoid receptor (GR), an immunocytochemical study was performed to investigate the intracellular localization of GR both in the presence or absence of ligand. With all fixation methods tested (paraformaldehyde, acetic acid in ethanol, Bouin's fixative, and bensochinone in PBS), it was possible to obtain specific GR staining. Fixation with paraformaldehyde was chosen for further studies on the effect of permeabilization, using several concentrations of Triton X-100 or saponin. A rat Rueber hepatoma (H-4-II-E) and a human uterus carcinoma (NHIK 3025) cell line were used as well as cultured hepatocytes from normal rat. The accessibility of the different cell compartments after fixation and permeabilization was tested for by using antibodies against cellular constituents with known locations (i.e. core-nucleosome proteins and tubulin), in combination with the anti-GR antibody in double immunofluorescence staining experiments. The specific GR stain ...

Ovarian thecal cells produce transforming growth factor-β which can regulate granulosa cell growth

Abstract: Ovarian thecal cells in culture were found to synthesize and secrete transforming growth factor-β (TGFβ). A component in thecal cell-conditioned medium was immunologically similar to TGFβ, as assessed with a RIA, and inhibited specific binding of TGFβ to its cell surface receptors. Thecal cell-secreted proteins also contained TGFβ biological activity, which was determined by stimulation of soft agar colony formation by AKR-2B indicator cells. Specific TGFβ antibodies precipitated a 25 K protein from radiolabeled thecal cell-secreted protein that comigrated with purified platelet-derived TGFβ. Both bovine thecal cell and rat thecal/interstitial cell preparations produced TGFβ, which required acid treatment to obtain fully active samples. The physiological significance of TGFβ production by thecal cells was addressed through an analysis of the effects of TGFβ on bovine granulosa cell growth. TGFβ inhibited epidermal growth factor stimulation of granulosa cell growth, but alone it had no apparent influence. ...

Regulation of Prolactin Production by Progestin, Estrogen, and Relaxin in Human Endometrial Stromal Cells

Abstract: Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).

A Comparative Study of Disaggregated Chick and Rat Osteoclasts in Vitro: Effects of Calcitonin and Prostaglandins*

Abstract: Disaggregated chick osteoclasts sedimented onto bovine cortical bone slices excavate deep and sharply defined resorption lacunae that stain intensely with toluidine blue. We have used this observation to develop a simple light microscopic method for quantifying the bone resorptive activity of chick osteoclasts in vitro. Using this technique, we have found that disaggregated chick osteoclasts are strikingly less sensitive than rat osteoclasts to salmon calcitonin (sCT). Bone resorption by rat osteoclasts was completely abolished by synthetic sCT at a concentration of 10 pg/ml. In contrast, sCT at concentrations up to 100 micrograms/ml did not significantly inhibit bone resorption by chick osteoclasts. Bone resorption by chick osteoclasts could, however, be inhibited by prostaglandin E2 at concentrations of 10(-6) M or more. In time-lapse video experiments, the motility of rat osteoclasts was rapidly inhibited by sCT (5-50 pg/ml), prostaglandins I2 and E2 (less than or equal to 10(-4) M), and Bu2cAMP (2.5 X 10(-4) M), whereas chick osteoclasts failed to show such a response. These findings suggest that CT does not play an important role in the regulation of osteoclastic activity in the chick and may explain why injection of the hormone has generally not been found to evoke an acute hypocalcemic response in birds. This fundamental difference in response to CT may limit the utility of chick osteoclast systems as models of mammalian bone resorption.

Retinol-induced stage synchronization in seminiferous tubules of the rat.

Abstract: Vitamin A deficiency (VAD) in rats causes a progressive germ cell depletion and cessation of spermatogenesis resulting in seminiferous tubules which contain only Sertoli cells, spermatogonia and a small number of preleptotene spermatocytes. Spermatogenesis can be rapidly restored by the administration of retinol. Our preliminary studies suggested a partial stage synchronization of many seminiferous tubules in VAD male rats subsequently treated with retinol. To confirm this observation and to achieve a better synchronization, VAD rats received 2 SC injections of retinol suspended in sesame oil followed by daily oral administrations of 0.5 mg of retinol. In all rats an almost perfect synchronous stage development of seminiferous tubules evolved in a predictable manner and was maintained through 2 spermiations.

Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells

Abstract: Collagenases that specifically cleave native collagen at neutral pH have been implicated in the maintenance and turnover of connective tissue. In bone, the origin of neutral collagenase has remained equivocal, although recent studies have indicated that it is synthesized by the osteoblast. In the present work, regulation of secretion of neutral collagenase and a collagenase inhibitory activity was investigated using the osteoblastic tumor cell line UMR 106-01 and a variety of bone-resorbing agents. Under basal conditions, UMR 106-01 cells produced very low levels of collagenase but substantial amounts of the inhibitory activity. Exposure to PTH and, to a lesser extent, 1,25-dihydroxyvitamin D3, prostaglandin E2, retinoic acid, and epidermal growth factor stimulated the release of collagenase, an effect not seen with interleukin-1 or heparin. The stimulation of collagenase by PTH was dose dependent, with a half-maximal response occurring at 10-8 M. Inclusion of isobutylmethylxanthine decreased the concentr...

Bovine Granulosa Cells Produce Basic Fibroblast Growth Factor

Abstract: Cultured bovine granulosa cells express the gene encoding basic fibroblast growth factor (bFGF). The bFGF gene is transcribed into 7.0- and 3.7-kilobase mRNA transcripts which are apparently translated into 16,000 mol wt bFGF-like growth factor. The granulosa cell-derived bFGF is bioactive, i.e. it can stimulate the proliferation of capillary endothelial or granulosa cells. This mitogenic effect is prevented by specific neutralizing anti-bFGF antibodies. Our results indicate that bFGF derived from granulosa cells can act as both autocrine and paracrine growth factor, and they further suggest that the factor may be involved in the development of the rich vasculature of the theca interna of the follicle.

Cholesterol Side-Chain Cleavage P450 Messenger Ribonucleic Acid: Evidence for Hormonal Regulation in Rat Ovarian Follicles and Constitutive Expression in Corpora Lutea*

Abstract: Using an affinity-purified antibody against cholesterol side-chain cleavage P450 (P450scc) and a human P450scc cDNA probe, 11 rat P450scc cDNA clones were identified and isolated from our rat granulosa cell λgt11 cDNA expression library. Two clones were plaque purified and subcloned into pBR322. One of these P450scc cDNA clones, approximately 1.2 kilobases (kb) in size, was used as a probe for Northern and filter hybridization assays to analyze the tissue distribution and hormonal regulation of P450scc mRNA in rat ovarian follicles and corpora lutea. Northern transfers revealed a single P450scc mRNA species about 2.0 kb in size. Filter hybridization assays showed that P450scc mRNA was low in granulosa cells and thecal cells of small antral follicles, was increased in both tissues of preovulatory follicles, and was rapidly (within 7 h) and maximally increased (30-fold) during hCG-induced luteinization. P450scc enzyme and mRNA were also elevated in corpora lutea isolated from pregnant rats (days 4–22 of ges...

Prostatic epidermal growth factor receptors and their regulation by androgens.

Abstract: Prostatic membranes contain high affinity [dissociation constant (KD) = 1.16 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I]iodo-EGF is specific since it is displaced by excess EGF but not by insulin, fibroblast growth factor, platelet-derived growth factor, or multiplication-stimulating activity. Affinity labeling with [125I]iodo-EGF and subsequent cross-linking with disuccinimidyl suberate demonstrated the specific binding of [125I]iodo-EGF to a macromolecule with a mol wt of 170,000. Castration of mature rats resulted in a 3- to 6-fold increase in [125I]iodo-EGF binding, while treatment of 7-day castrated rats with 5 alpha-dihydrotestosterone decreased the number of binding sites. Administration of estrogen or progesterone produced a slight decrease in EGF binding sites but not nearly to the extent observed with 5 alpha-dihydrotestosterone, suggesting that the observed effect is androgen specific. These results demonstrate that rat prostate contains specific binding sites for EGF and that their level is modulated by androgens.

Concomitant Effects of Growth Hormone on Secretion of Insulin-Like Growth Factor I and Progesterone by Cultured Porcine Granulosa Cells*

Abstract: The observation that GH deficiency delays the onset of puberty has raised the question of the effect of GH on gonadal development. In addition, recent studies in the rat have indicated that GH is able to elevate ovarian levels of immunoreactive insulin-like growth factor I (iIGF-I) in vivo and enhance FSH-induced granulosa cell differentiation in vitro. To evaluate further the possibility of direct effects of GH on ovarian function, we have studied the action of GH on the secretion of iIGF-I and progesterone by cultured porcine granulosa cells from immature follicles. The effects of GH were compared with those of estradiol (E2) and FSH, hormones previously shown to stimulate steroidogenesis and iIGF-I production in this system. GH-stimulated cultures secreted 7.8 times as much iIGF-I per cell as control cultures, while cultures treated with E2 plus FSH secreted 4.5 times as much, and the combination of all three hormones produced an additional increment. The GH-dependent immunoreactivity was localized to two peaks on gel filtration which coeluted with authentic IGF-I and with an IGF-binding protein. In contrast to the results with iIGF-I secretion, GH was a relatively ineffective stimulator of progesterone secretion, resulting in levels 2.6 times the control value, compared to levels 7.4-fold the control value in cultures treated with E2 plus FSH. However, when the three agonists were combined, a synergistic interaction was observed which resulted in progesterone values 33.3 times the control value. In parallel studies, PRL was unable to mimic the effects of GH on iIGF-I or progesterone secretion. In summary, GH has direct stimulatory actions on porcine granulosa cells. Compared to E2 and FSH, established stimulators of these cells, GH is at least comparable in effectiveness with regard to iIGF-I secretion, but less effective as a stimulator of steroidogenesis. However, GH dramatically enhances the effects of E2 and FSH on progesterone secretion. These effects of GH could be important during the onset of puberty, when GH levels in plasma are elevated.

Formation of multinucleated cells that respond to osteotropic hormones in long term human bone marrow cultures.

Abstract: Studies of osteoclasts and their precursors in normal and pathological states have been severely hampered by the lack of an in vitro system for forming osteoclasts. We developed a human marrow culture system in which multinucleated cells with several characteristics of osteoclasts form. Multinucleated cells began to form during the first week of culture, with maximum numbers formed after 3 weeks. PTH (25–50 ng/ml) and 1,25-dihydroxyvitamin D3 (10-10–10-8M) increased formation of these cells, and these effects were inhibited by calcitonin. These multinucleated cells contained nonspecific esterase and tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts, and had several ultrastructural features of osteoclasts. We used this marrow cell culture technique to study a patient with hyperparathyroidism and markedly increased osteoclasts on bone marrow biopsy. The marrow from this patient formed increased numbers of multinucleated cells in vitro. After parathyroidectomy both multinucleated cell forma...