bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic effects of progesterone and oestradiol on rat LH subunit mRNA
Authors: Corbani M. , Counis R. , Wolinska-Witort E. , d'Angelo-Bernard G. , Moumni M. , Jutisz M.
Published: December 2008

The effects of oestradiol and progesterone on LH-subunit mRNA levels were investigated in ovariectomized rats. Four weeks after ovariectomy, rats were implanted with silicone elastomer capsules containing oestradiol and/or injected daily with progesterone in oil (5 mg/rat) for 8 days. The levels of pituitary mRNA encoding α and LH-β were determined using direct hybridization with specific [32P]cDNA probes. After oestradiol implantation in ovariectomized rats, both α and LH-β mRNA decreased with time, with maximum inhibition after 6–8 days of treatment. Progesterone injected alone did not show any effect on α and LH-β mRNA. Cytosolic progesterone receptors, determined using [3H]methyl-17α-progesterone as ligand, were undectable in control ovariectomized rats. In contrast, 2 days after oestradiol implantation, the number of receptors increased to 287·5 ± 35·4 (s.e.m.) fmol/pituitary and reached a plateau of 400 ± 21·8 fmol/pituitary after 4 days. The effects of progesterone were therefore examined by first implanting ovariectomized rats with oestradiol to induce progesterone receptors and then injecting progesterone daily for a further period of 6 days. As a result of this treatment, progesterone induced a decrease in the pituitary gland contents of both α and LH-β mRNAs, and LH release was significantly greater than that observed in the group receiving oestradiol alone. Moreover, the mRNA levels in the animals treated with oestradiol plus progesterone were lower after 8 days of treatment than those observed in ovariectomized rats treated with a tenfold higher dose of oestradiol alone.

These data demonstrate that progesterone, together with oestradiol, is capable of negatively regulating the mRNAs encoding subunits in vivo, provided that progesterone receptors are present in the pituitary gland.
Transmembrane Na+/H+ exchange in the rat thyroid cell strain FRTL-5: a possible role in insulin-like growth factor-I-mediated proliferation
Authors: Woods D. J. , Soden J. , Tomlinson S. , Bidey S. P.
Published: December 2008

Using the fluorescent pH indicator 2′7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein to monitor intracellular pH (pHi), we have investigated whether transmembrane Na+/H+ exchange, as measured by experimental changes in pHi under bicarbonate-free incubation conditions, may be involved in the early growth-promoting actions of insulin-like growth factor-I (IGF-I) on the rat thyroid cell stain FRTL-5. In initial studies to characterize Na+/H+ exchange in FRTL-5 cell suspensions, the recovery of a resting pHi in acid-loaded cells was shown to be dependent upon the presence of extracellular Na+, was enhanced by the presence of the sodium ionophore monensin and was abolished by amiloride, an antagonist of Na+/H+ antiport activity.

Unlike TSH, which was without effect on the pHi of FRTL-5 cells for up to 15 min after addition, IGF-I (1000 μg/1) caused a rapid and sustained increase within 3 min, which was abolished in medium in which Na+ had been replaced with an iso-osmotic level of choline chloride. The change in pHi in response to IGF-I was mimicked by phorbol 12-myristate 13-acetate (PMA; 100 nmol/1), an activator of thyroid cell proliferation. In the presence of TSH, exposure of cells to IGF-I or PMA had no additional effect on the cytoplasmic alkalinization induced by either of these two agonists alone. However, blockade of transmembrane Na+/H+ exchange with amiloride inhibited both the individual actions of IGF-I and PMA on [methyl-3H]thymidine incorporation, and the synergistic interaction between TSH and IGF-I. These findings are consistent with a differential mode of action of TSH and IGF-I on the early events associated with FRTL-5 cell proliferation, and suggest involvement of an amiloride-sensitive transmembrane Na+/H+ exchange in mediating the early cellular response to the latter. Furthermore, the later actions of PMA, which are also dependent upon early transmembrane Na+/H+ exchange, differ from those of IGF-I with respect to interaction with the growth-promoting actions of TSH.
Thyrotrophin regulation of apical and basal exocytosis of thyroglobulin by porcine thyroid monolayers
Authors: Chambard M. , Depetris D. , Gruffat D. , Gonzalvez S. , Mauchamp J. , Chabaud O.
Published: December 2008

Exocytosis, the ultimate step in thyroglobulin secretion, has been studied in porcine thyroid cells cultured in monolayers on the permeable bottom of culture chambers. We have previously demonstrated, using this culture system, that apical secretion accounts for 85–95% of total secretion of newly synthesized thyroglobulin. When cells were cultured for several days with bovine TSH (25 μU/ml) in the basal medium, the rate of glycoprotein accumulation in the upper compartment was three times higher than that in the absence of TSH. In contrast, the rate of thyroglobulin released into the basal medium (5–15% of total secreted thyroglobulin) appeared unmodified by chronic TSH stimulation. To investigate the effect of acute TSH stimulation on thyroglobulin exocytosis in the apical and basal compartments, pulse-chase experiments were carried out with the same culture system. The release of radiolabelled thyroglobulin (1·5-h pulse) into the apical medium was increased threefold during the 2-h chase period under TSH stimulation. The radiolabelled thyroglobulin released into the basal medium was increased only 1·5- to 2-fold, and stimulation disappeared after 1 h. The effect of TSH was maximal when the chase medium contained 50 μU TSH/ml. However, cells cultured for several days in the presence of 25 μU TSH/ml before the pulse-chase experiment, appeared desensitized to acute TSH stimulation. Similar responses were observed when the chase medium contained 8-chloro-cyclic AMP or cholera toxin. This study provides another example of the pleiotropic effect of TSH, mediated by cyclic AMP, on the sequential steps of thyroglobulin gene expression in cultured thyroid cells in which the polar character of the epithelial cells is well preserved.
Evidence for differential effects of noradrenaline and somatostatin on intracellular messenger systems in rat islets of Langerhans
Authors: Hurst R. D. , Morgan N. G.
Published: December 2008

The mechanisms involved in inhibition of insulin secretion by somatostatin and noradrenaline were compared in order to establish whether the receptors for these agents are coupled to similar effector systems in the pancreatic B cell. Both agents significantly reduced forskolin-induced adenylate cyclase activity in islet homogenates, although noradrenaline was more effective than somatostatin. The capacity of noradrenaline to inhibit insulin secretion was largely unaffected by agents that increase intracellular cyclic AMP, whereas the effect of somatostatin as an inhibitor was markedly reduced under these conditions. Both noradrenaline and somatostatin inhibited the stimulation of insulin secretion induced by K+ depolarization, but different mechanism were involved. Somatostatin significantly inhibited K+-stimulated 45Ca2+ efflux and influx in islets, while noradrenaline exerted only a minor influence on these processes. The data indicate that noradrenaline controls insulin secretion by a mechanism which operates beyond the level of intracellular messenger generation. In contrast, somatostatin exerts at least part of its inhibitory effect on insulin secretion by directly controlling islet cell Ca2+ influx in a manner which may be regulated by cyclic AMP.
Activation of mouse osteoblast growth hormone receptor: c-fos oncogene expression independent of phosphoinositide breakdown and cyclic AMP
Authors: Slootweg M. C. , van Genesen S. T. , Otte A. P. , Duursma S. A. , Kruijer W.
Published: December 2008

Addition of human GH (hGH) to primary mouse osteoblasts resulted in rapid and transient induction of the c-fos and c-myc proto-oncogenes and preceded hGH-induced mitogenesis. Human GH-induced c-fos expression was maximal after 30 min, resulting in a 10- to 15-fold increase over unstimulated cells, and returned to prestimulation levels within 60 min of the addition of hGH. Induction of the c-fos gene by hGH was dose dependent and also occurred in the absence of protein synthesis, resulting in superinduction of the c-fos gene. The induction of the c-fos gene by hGH was mediated by a somatotrophic (GH) rather than a lactogenic (prolactin) receptor on primary mouse osteoblasts, as indicated by a 10- to 100-fold greater potency of hGH compared with ovine prolactin in stimulating the expression of the c-fos gene. Primary mouse osteoblasts also induced the c-fos gene in response to epidermal growth factor, insulin-like growth factor-I and several agents, including phorbol 12-myristate 13-acetate (TPA), forskolin and A23187, that are known to activate signal transduction pathways involved in the action of growth factors.

Addition of hGH to primary mouse osteoblasts did not result in increased phosphoinositide breakdown, while selective deactivation of the diacylglycerol—protein kinase C and inositol 1,4,5—trisphosphate—Ca2+ pathways by long-term TPA pretreatment or depleting intracellular Ca2+ stores had no effect on hGH-induced c-fos expression. Human GH did not alter basal cyclic AMP levels in mouse osteoblasts. The immediate consequences of GH—receptor interaction as well as the mechanism of signal transduction leading to induction of the c-fos gene remain, therefore, unresolved.
Pregnancy-blocking progesterone antibody targets specifically the uterus through its progesterone-binding sites
Authors: Wang M.-W. , Whyte A. , Heap R. B. , Taussig M. J.
Published: December 2008

Passive immunization with a mouse monoclonal antibody against progesterone, designated DB3, blocks pregnancy in several species. We have previously reported that DB3 localizes in the mouse uterine epithelium shortly before normal implantation. This phenomenon is pregnancy dependent and specific for the progesterone antibody. In this study we demonstrate that DB3 is present in the lumen of the uterus 36 h after an i.p. injection; this correlates with the time of maximum antibody reaction on the uterine epithelium. Incubation of DB3 with free progesterone, progesterone-hemisuccinate or progesterone—bovine serum albumin before administration prevented its localization on the epithelium, indicating that the localization requires free progesterone-binding sites and thus probably depends upon progesterone binding. In addition, studies in vitro show that DB3 can effectively bind to progesterone carried by high-affinity progesterone-binding protein purified from coypu plasma. We suggest that specific targeting of DB3 may be through progesterone associated with a progesterone-binding molecule on the membrane of the uterine epithelia. This may be an important part of the mechanism of antibody action against implantation.
Regulation of α and thyrotrophin-β subunit mRNA levels by androgens in the female rat
Authors: Ahlquist J. A. O. , Franklyn J. A. , Ramsden D. B. , Sheppard M. C.
Published: December 2008

Thyroid and steroid hormones act by similar mechanisms to influence gene expression in the anterior pituitary gland. The genes encoding the common α and TSH-β glycoprotein subunits are known to be regulated by thyroid hormones; we report here the effects of androgen administration on levels of α and TSH-β mRNA in pituitary cytoplasm in the euthyroid and hypothyroid female rat. Dihydrotestosterone (DHT) suppressed both α and TSH-β mRNAs to levels lower than those found in untreated animals; a similar reduction was seen in hypothyroid animals treated with DHT. A biphasic response of TSH-β mRNA was seen following administration of tri-iodothyronine (T3) to hypothyroid rats, with early stimulation followed by later inhibition; these changes were also evident after administration of T3 to androgen-treated animals, although mRNA levels were again suppressed. The effects of testosterone were similar to those of DHT. In contrast to the changes in mRNA levels, androgen administration did not lead to significant alterations in serum TSH concentrations or pituitary TSH content.

These results indicate that, like thyroid hormones, androgens suppress both α and TSH-β subunit mRNA levels in the female rat. Androgens, however, exert differential effects on TSH synthesis and release which contrast with those of thyroid hormones.
Influences of photoperiod on sexual behaviour, neuroendocrine steroid receptors and adenohypophysial hormone secretion and gene expression in female golden hamsters
Authors: Bittman E. L. , Hegarty C. M. , Layden M. Q. , Jonassen J. A.
Published: December 2008

Exposure to short daylengths arrests the oestrous cycle, provokes daily gonadotrophin surges and reduces the ability of exogenous oestradiol to trigger behavioural receptivity in golden hamsters. In order to examine neuroendocrine effects of photoperiod which might underlie these responses, ovariectomized hamsters were maintained under long or short photoperiods for 54 days before treatment with cholesterol or various doses of oestradiol-17β. Short days reduced the ability of low doses of oestrogen to prime hamsters for the induction of oestrus by progesterone. Upon repetition of oestrogen priming 2 weeks later, photoperiod was without significant influence on the concentrations of nuclear oestrogen receptors or cytosolic progestin receptors in a block of tissue containing the hypothalamus and preoptic area.

Oestradiol treatment provoked significant increases in serum concentrations of LH and prolactin in the afternoon, but photoperiod did not alter the positive-feedback efficacy of this gonadal steroid hormone. Adenohypophysial LH-β subunit and prolactin mRNAs were suppressed by short days in ovariectomized hamsters not treated with oestradiol. Oestradiol decreased expression of the LH-β subunit gene in both stimulatory and inhibitory photoperiods, but increased prolactin mRNA abundance in both long and short days. Photoperiod therefore exerts pronounced steroid-independent effects on phasic LH and prolactin secretion, but regulation of adenohypophysial abundance of LH-β subunit and prolactin mRNAs by oestradiol is not markedly influenced by daylength. Photoperiodic regulation of the priming effects of oestradiol on behavioural receptivity may result from modulation of events occurring subsequent to steroid—receptor interactions, or involve changes in receptor populations not detectable by the present methods.
Effect of glucocorticoids on α1-adrenergic receptor binding in rat vascular smooth muscle
Authors: Haigh R. M. , Jones C. T.
Published: December 2008

Glucocorticoids are known to have marked effects on blood pressure regulation, predominantly through altering cardiovascular sensitivity to noradrenaline. However, the molecular mechanisms underlying this action remain unclear. As part of our studies into these we have measured α1-adrenergic receptor binding using the ligand [3H]prazosin in plasma membrane fractions of aortas prepared from control, adrenalectomized and dexamethasone-treated adrenalectomized rats. In controls there were 50±8 (s.e.m.; n=6) fmol α1-adrenergic receptors/mg membrane protein (Bmax) with a dissociation constant (Kd) of 0·52±0·10 nm (n=6). Adrenalectomy 8 days before tissue preparation caused a 40% decrease in Bmax and a 60% decrease in Kd. Dexamethasone replacement after adrenalectomy returned these values close to those of controls. Noradrenaline competed for the [3H]prazosin-binding sites. Computer analysis by a non-linear curve-fitting program (LIGAND) showed that noradrenaline binding was to a heterogeneous population of high- and low-affinity receptors with Kd values of 1·87±0·73 μm and 0·48±0·12 mm (n=5) respectively. Guanosine thiotriphosphate (GTP[S]) caused the conversion of high-affinity to low-affinity binding, consistent with the model of the high-affinity sites being coupled to a G protein. After adrenalectomy, noradrenaline binding was to a homogeneous population of low-affinity receptors; hence, the effect of GTP[S] was no longer apparent, suggesting that under these conditions the α1-adrenergic receptors were unable to couple to a G protein. The two-site model of binding and GTP[S] effect was returned by dexamethasone treatment. These data provide evidence that glucocorticoids not only modulate the number of α1-adrenergic receptors on vascular smooth muscle, but also cause disruptions in receptor—G protein coupling. This may be an important mechanism by which glucocorticoids exert their effect on cardiovascular sensitivity.
Vasopressin stimulates cortisol secretion and phosphoinositide catabolism in cultured bovine adrenal fasciculata/reticularis cells
Authors: Bird I. M. , Nicol M. , Williams B. C. , Walker S. W.
Published: December 2008

Cells isolated from the zona fasciculata/reticularis (ZFR) of the bovine adrenal cortex and maintained in culture were found to secrete cortisol in response to vasopressin stimulation. The increased cortisol secretion was dose dependent, with a threshold response at 1 nm and a maximal response (1·68-fold over basal) at 0·1 μm. In cells cultured in the presence of [3H]inositol (to prelabel the membrane phosphoinositide pool), stimulation with vasopressin in the presence of LiCl (10 mm) resulted in a similar dose-dependent increase in labelling of the phosphoinositol fraction, with a maximal response (1·45-fold over basal) at 10 nm. The increased labelling of the phosphoinositol fraction was independent of extracellular Ca2+ as it was not abolished in medium with [Ca2+ ] buffered to intracellular resting levels. This suggests that vasopressin stimulation results in the activation of a phosphoinositidase C. It is probable that cortisol secretion by bovine ZFR cells in response to vasopressin is dependent upon activation of this Ca2+-independent phosphoinositidase C. However, the small magnitude of the cortisol secretory response makes it unlikely that vasopressin is a primary regulator of cortisol secretion in vivo.
The gene for the β subunit of porcine FSH: absence of consensus oestrogen-responsive element and presence of retroposons
Authors: Hirai T. , Takikawa H. , Kato Y.
Published: December 2008

To elucidate the structure and control of expression of the porcine FSH-β subunit gene, two genomic clones were isolated and the entire gene structure was determined to the extent of 10 kb, consisting of 6 kb of the 5′-flanking region and 4 kb of the transcriptional unit. The porcine FSH-β gene consisted of three exons the same as the human and bovine genes, but the positions of both splicing sites of porcine intron-1 were unique. It is known that the synthesis of FSH is regulated by gonadal steroids, gonadotrophin-releasing hormone (GnRH) and inhibin. However, the consensus steroid-responsive element was unexpectedly absent in the 5′-flanking region of 6 kb. On the other hand, the potential binding sites for activator protein-1 (AP1) and AP2, which might be stimulated by the GnRH—protein kinase C cascade, were present at seven and five positions respectively. An imperfect cyclic AMP-responsive element was also present. Southern blot analyses, using the cDNA and genomic fragments as probes, gave smear patterns suggesting the presence of repetitive sequences in the porcine FSH-β gene. A survey of homology with the repetitive sequences revealed that short interspersed repeated sequences (SINES)-type non-viral retroposons were present with about 250 bp length repeats twice in the 5′-flanking region and once each in intron-1 and the 3′-flanking region. Other SINES-like sequences were also found in intron-1, exon-2 and exon-3. In comparison with the 5′-flanking sequences of the porcine α and LH-β genes, there were no significantly conserved regions, implying a lack of common modulation of the three subunit genes.
The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety
Authors: Castro M. G. , Lowenstein P. R. , Saphier P. W. , Linton E. A. , Lowry P. J.
Published: December 2008

We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to β-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1–41) and CRH(36–41). They did not cross-react with the CRH antagonist CRH(9–41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1–41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human preproCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1–41). The results indicate that these antibodies can recognize CRH(1–41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.
Depletion of islet amyloid polypeptide in the spontaneously diabetic (BB) Wistar rat
Authors: Bretherton-Watt D. , Ghatei M. A. , Legon S. , Jamal H. , Suda K. , Bloom S. R.
Published: December 2008

Islet amyloid polypeptide (IAPP) in the pancreas of the spontaneously diabetic (BB) Wistar rat was examined by radioimmunoassay, and IAPP mRNA levels were determined by Northern blotting. IAPP-like immunoreactivity in the diabetic rat pancreas was found to be significantly depleted compared with control (non-diabetic) BB rats (85·9±5 pmol/g in control rats, n = 8, vs 8·97 ± 0·9 pmol/g in diabetic rats, n=5; mean ± s.e.m.). A similar change in insulin concentrations was found, although insulin was present in approximately 100-fold greater amounts than IAPP. Chromatography of the IAPP immunoreactivity revealed a single molecular form, corresponding to synthetic IAPP. Northern blot analysis of pancreatic RNA (n = 4) revealed that IAPP mRNA in the diabetic group was depleted to 22% of the signal intensity in the control group. Insulin mRNA was dramatically reduced to only 4% of the control group and, in contrast, somatostatin was relatively unaffected, with the diabetic group retaining 86% of signal compared with the controls.

This animal model of insulin-dependent diabetes results from severe autoimmune destruction of the β cell. The extremely low levels of both insulin and its messenger RNA are in agreement with this. These results demonstrate that this pathological state is also associated with a loss of IAPP from the pancreas. Insulin-dependent diabetes is associated with a range of metabolic disturbances. It is possible that the concomitant depletion of IAPP may be a contributory factor in exacerbating the condition.
Variable methylation of the 5′-flanking DNA of the human pro-opiomelanocortin gene
Authors: Lavender P. , Clark A. J. L. , Besser G. M. , Rees L. H.
Published: December 2008

The pro-opiomelanocortin gene is widely expressed in human tissues, although both transcriptional initiation sites and regulation appear to be tissue specific. In order to determine how promoter and enhancer choice is effected, we have studied the methylation pattern of the gene in a number of normal tissues, tumours and cell lines. Variability of this pattern was observed in the 5′-flanking DNA, particularly at the HpaII site located at −304 bp upstream from the pituitary CAP site. This site was generally methylated in tissues likely to express the predominant extrapituitary (800 nucleotide) message, while in tissues known to express the normal pituitary (1150 nucleotide) message and longer species, a tendency towards undermethylation was observed. Although the sites at which variable methylation occurs did not correspond to established binding sites for regulatory proteins, many of these regions remain to be determined and thus it is possible that methylation may be influential in the tissue-specific regulation of this gene.
Maitotoxin increases inositol phosphates in rat anterior pituitary cells
Authors: Sortino M. A. , Delahunty T. M. , Yasumoto T. , Cronin M. J.
Published: December 2008

Maitotoxin is a potent marine poison that mobilizes calcium in most vertebrate cell types and accelerates secretion from anterior pituitary cells. It is not known whether voltage-sensitive calcium channels or other mechanisms initiate the effects of maitotoxin on anterior pituitary cells. Changes in intracellular Ca2+ levels may also be achieved by releasing internal calcium stores via inositol trisphosphate (InsP3). Indeed, maitotoxin rapidly increased inositol phosphate accumulation in a concentration-dependent manner. Calcium channel antagonists such as nifedipine and verapamil did not block this response nor did calcium-mobilizing agents (BAYk8644, A23187) mimic this effect. These data suggest that the mechanism by which maitotoxin acts at the pituitary may include the activation of an enzyme that produces the calcium-mobilizing signal InsP3.
EVIDENCE FOR A PREVIOUSLY UNIDENTIFIED UPSTREAM EXON IN THE HUMAN OESTROGEN RECEPTOR GENE.
Authors: Keaveney M. , Klug J. , Dawson M.T. , Nestor P.V. , Neilan J.G. , Forde R.C. , Gannon F.
Published: December 2008

The presence of a previously unidentified exon upstream of the originally described human oestrogen receptor (hOR) gene is demonstrated. This is shown to be spliced to the 5′ untranslated region of the previously designated exon I. The resulting genomic structure of the human gene is thus in agreement with the structure of the mouse OR gene and highlights the conservation of an 18 amino acid upstream open-reading frame formed from the above splicing event. Taken in conjunction with previous publications this would suggest that the hOR gene is a complex transcriptional unit that contains two promoters.
Regulation by progesterone of the high-affinity state of myometrial β-adrenergic receptor and of adenylate cyclase activity in the pregnant rat
Authors: Cohen-Tannoudji J. , Vivat V. , Heilmann J. , Legrand C. , Maltier J. P.
Published: December 2008

The effects of pregnancy or progesterone dominance on the β-adrenergic responsiveness of the uterus were studied in myometrial membranes from mid-and late-pregnant rats (day 15 and on the 16th h of day 22 of pregnancy respectively) or 24 h after administration of progesterone. Levels of the high (RH)- and low (RL)-affinity states of the β-adrenergic receptor were determined by competition experiments between 125I-labelled cyanopindolol binding and the selective β-agonist isoproterenol. The ratio KL/KH (respective dissociation constants) was determined since it also reflects the degree of formation of the high-affinity state of the β-adrenergic receptor. From day 15 to the 10th h of day 22 of pregnancy, two distinct affinity states were apparent: 80–55% RH (KH=0·31–0·21 μm) and 45–20% RL (KL=14–5 μm) with a ratio of KL/KH of 55–34. In the last 6 h before birth, β-adrenergic receptors underwent uncoupling which was paralleled by decreased responsiveness of myometrial adenylate cyclase to isoproterenol (maximum velocity (Vmax)=17±3 vs 44±3 fmol cyclic AMP/10 min per mg protein on day 15). At this stage of pregnancy, previous exposure to progesterone resulted in a 1·8-fold increase in 125I-labelled cyanopindolol-binding sites (Bmax) and the reappearance of the high-affinity state (67% RH, KH=0·19±0·04 (s.e.m.) μm, ratio KL/KH=81·1 ± 16·9). These results were reversed in the presence of the antiprogestin RU486 (100% RL, KL=24·6±4·1 μm, 41% reduction of Bmax). Moreover, after progesterone, adenylate cyclase activity was strongly stimulated by isoproterenol (Vmax=60±12 fmol cyclic AMP/10 min per mg protein vs 17±3 in controls). The data suggest (1) that progesterone may exert a permissive effect on β-adrenergic responsiveness of the pregnant rat myometrium and (2) that at term, both a desensitization mechanism involving uncoupling of β-adrenergic receptors and a decrease in activation of adenylate cyclase lead to a loss of myometrial response to β-agonists.
Predicted structure of the bovine calcitonin gene-related peptide and the carboxy-terminal flanking peptide of bovine calcitonin precursor
Authors: Collyear K. , Girgis S. I. , Saunders G. , MacIntyre I. , Holt G.
Published: December 2008

We have isolated from a bovine genomic library a clone which contains the calcitonin (CT) and CT gene-related peptide (CGRP) sequences, using probes representing the human CT and CGRP sequences. Sequence analysis has identified the nucleotide sequence coding for bovine CT, its C-terminal flanking peptide and bovine CGRP. The deduced amino acid sequence of bovine CGRP revealed a significant homology with other CGRPs so far reported. It differs by only one amino acid from rat CGRPα and porcine CGRP, and by three and four amino acids from human CGRPβ and α respectively. Bovine CT has, however, only 14 out of 32 residues in common with human CT. As in the human CT precursor, the C-terminal flanking peptide of bovine CT precursor is a 21 amino acid peptide. It shares only 11 residues in common with its human counterpart. This study thus provides further evidence that CGRP, in contrast to CT and its C-terminal flanking peptide, is a highly conserved molecule.
Self-inhibition of steroid secretion by amphibian adrenocortical cells is not mediated through glucocorticoid receptors
Authors: Netchitailo P. , Larcher A. , Leboulenger F. , Feuilloley M. , Philibert D. , Vaudry H.
Published: December 2008

To investigate a possible direct action of glucocorticoids on adrenal steroidogenesis, the effect of corticosterone on the conversion of pregnenolone into various metabolites by frog adrenal tissue was examined. Frog interrenal slices were incubated with [3H]pregnenolone (1 mCi/ml) and the various labelled metabolites analysed by reverse-phase high-performance liquid chromatography. With the methanol gradient used, five identified steroids were resolved: progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone. Corticosterone (10 μg/ml) induced a 45–80% decrease in all steroids synthesized from [3H]pregnenolone. In contrast, the glucocorticoid agonist dexamethasone did not reduce the rate of conversion of pregnenolone into its metabolites. In addition, the inhibitory effect of corticosterone was not reversed by the specific glucocorticoid antagonist RU 43044. These results show that corticosterone exerts a direct inhibitory effect on adrenal steroid secretion. In addition, our data indicate that the ultra-short regulation induced by corticosterone is not mediated through glucocorticoid receptors.
Stimulation of an individual cell with peptide hormone in a prescribed region of its plasma membrane results in a compartmentalized cyclic AMP-dependent protein kinase response
Authors: Podesta E. J. , Solano A. R. , Lemos J. R.
Published: December 2008

This work describes the stimulation by a peptide hormone of an individual cell in a prescribed region of its plasma membrane. When Leydig cells were stimulated via a section of membrane tightly sealed to an electrode containing LH, a very localized area exhibited the morphological change known as 'rounding up', which is a cyclic AMP-dependent protein kinase-mediated response. This localized stimulation did not produce a wider response through intracellular, intermembranous or extra-cellular signals. Each individual cell responded to peptide stimulation gradually, with an increase over time and with dose. In contrast, when the stimulation was accomplished using a non-hydrolysable cyclic AMP analogue in the patch electrode, a general response throughout an individual cell was produced. Locally stimulated peptide hormone receptors, adenylate cyclases and cyclic AMP-dependent protein kinases appear to be closely associated so that second messenger production and the effects it mediates are compartmentalized.
Interleukin-1β production in vivo and in vitro in rats and mice measured using specific immunoradiometric assays
Authors: Bristow A. F. , Mosley K. , Poole S.
Published: December 2008

Activated cells of the monocyte—macrophage lineage produce two forms of the inflammatory cytokine interleukin-1 (IL-1), IL-1α and IL-1β, of which IL-1β is the predominant secreted form and has a wide range of modulatory effects on the endocrine system. Immunoassays of human IL-1β have been described, but are not suitable for measurement of rat and mouse IL-1β because of limited cross-reactivity. Polyclonal sheep anti-rat or sheep anti-mouse IL-1β antisera were used to develop sensitive and specific immunoradiometric assays for rat and mouse IL-1β. Secretion of IL-1β from endotoxin-activated monocytes or macrophages was measured in vitro or in vivo in both species. In vitro, rat monocytes and mouse macrophages produced IL-1β in response to endotoxin, with a relatively small proportion of total IL-1β being secreted. In vivo, endotoxin stimulated an increase in plasma IL-1β in both animals. The development of these assays will facilitate studies of the role of endogenous IL-1β in animal endocrine models.
Regulation of the type-II gonadotrophin α and β subunit mRNAs by œstradiol and testosterone in the European eel
Authors: Quérat B. , Hardy A. , Fontaine Y.A.
Published: December 2008

The gonadotrophic function of the European eel (Anguilla anguilla L.) at the silver stage is very weak: gonadotrophin-releasing hormone (GnRH) secretion is deficient and, moreover, dopamine inhibition overrides GnRH action. At the silver stage, œstradiol stimulates the biosynthesis of the type-II gonadotrophin (GTH-II). To study the molecular mechanism of this activation further, we examined the effect of testosterone and œstradiol administration on pituitary levels of mRNA encoding GTH-II α and β subunits. Corresponding eel cDNA probes and Northern blot analysis were used. After 2 weeks, testosterone and œstradiol implantation resulted in a strong increase in mRNA encoding the GTH-II β subunit (7-fold and 25-fold, respectively) and in a slight, but non-significant, rise in the a subunit mRNA level (1.8-fold and 1.5-fold, respectively). Co-implantation of these two steroids suggested a potentiation of their effects on the β subunit (104-fold) while an additive effect was indicated on the α mRNA level (2.9-fold). Effects were detectable within 4 days and were maximal 4 weeks after implantation. These results indicate that in the European eel at the silver stage, gonadal steroids stimulate differentially the expression of GTH-II subunit genes at a pretranslational level.
The dopamine D2 receptor is expressed in GH3 cells
Authors: Johnston J. M. , Wood D. F. , Bolaji E. A. , Johnston D. G.
Published: December 2008

Some pituitary tumours respond to dopamine by decreasing the release of prolactin and/or GH and by inhibition of tumour growth. Certain tumours are unresponsive. Dopamine D2 receptor high-affinity binding is impaired in these tumours, and the rat GH3 cell line behaves in a similar way. The hypothesis that the dopamine-binding defect results from impaired D2 receptor gene expression has been tested in the present study. On Northern blots, D2 receptor mRNA was present in both normal rat pituitary cells and in GH3 cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis identified a putative D2 receptor protein in normal and GH3 cell membranes. The lack of effect of dopamine in GH3 cells does not reflect the absence of D2 receptor gene expression.
Studies on an adherent variant of the human promyelomonocytic HL60 leukaemia cell line that constitutively expresses 25-hydroxyvitamin D3-24-hydroxylase activity which is inhibited by 1α,25-dihydroxyvitamin D3
Authors: Hayes M. E. , Bayley D. , Mawer E. B.
Published: December 2008

The constitutive expression of 25-hydroxyvitamin D3-24-hydroxylase (25-(OH)D3-24-hydroxylase) activity has been studied in an adherent variant (Ad-HL60) of the human promyelomonocytic leukaemia cell line HL60. The Ad-HL60 cells have a more differentiated phenotype than the non-adherent cells from which they were derived, and synthesized 1.88±0.07 (±s.e.m.) pmol 24,25-(OH)2D3/h per 106 cells following culture in RPMI-1640 medium containing <0.02 nm 1α,25-(OH)2D3. They also synthesized 1.66±0.05 pmol 24,25-(OH)2D3/h per 106 cells following culture in 1α,25-(OH)2D3-free medium supplemented with 1 g bovine serum albumin/l instead of 10% serum. In contrast, non-adherent HL60 cells required exposure to 10–100 nm 1α,25-(OH)2D3 to induce equivalent 24,25-(OH)2D3 synthesis. The 25-(OH)D3-24-hydroxylase expressed by Ad-HL60 cells had an apparent Michaelis constant of 1 μm and maximal rate of 20 pmol/h per 106 cells with substrate concentrations from 0.012 to 1.2 μm/incubation (5–500ng/ml). Furthermore, 24,25-(OH)2D3 synthesis was inhibited in a dose-dependent manner by ketoconazole (0.01–10 μm), suggesting that the enzyme is cytochrome P-450 dependent.

Ad-HL60 cells expressed approximately 3500 specific receptors for 1α,25-(OH)2D3/cell with a dissociation constant of 40 pm. Following exposure to 0.1–100 nm 1α,25-(OH)2D3, Ad-HL60 cell proliferation was significantly inhibited compared with controls grown in medium containing <0.02 nm 1α,25-(OH)2D3 for 96h. Expression of 25-(OH)D3-24-hydroxylase was also inhibited in a dose- and time-dependent manner; however, expression of non-specific esterase was not induced. Both of these findings are contrary to those previously demonstrated for non-adherent HL60 cells, whereas the dose-dependent inhibition of cell proliferation by 1α,25-(OH)2D3 occurs in both adherent and non-adherent phenotypes. These observations on Ad-HL60 cells represent the first description of a cell type in which 1α,25-(OH)2D3 appears to inhibit 25-(OH)D3-24-hydroxylase activity. The Ad-HL60 cells also constitutively metabolized 1α,25-(OH)2D3 in a manner consistent with formation of 1α,24,25-(OH)3D3 without previous exposure to 1α,25-(OH)2D3. In contrast, many other cell types, including non-adherent HL60 cells, require exposure to 1α,25-(OH)2D3 to induce metabolism of 1α,25-(OH)2D3 to 1α,24,25-(OH)3D3, a reaction that represents the initial step for catabolism of 1α,25-(OH)2D3 to calcitroic acid.
Quantitative comparison of insulin-like growth factor mRNA levels in human and rat tissues analysed by a solution hybridization assay
Authors: Möller C. , Arner P. , Sonnenfeld T. , Norstedt G.
Published: December 2008

In this study a solution hybridization assay was evaluated for its application to the measurement of levels of specific mRNAs. The evaluation included parameters such as incubation time, hybridization stringency and probe concentration/structure. Both short (50 bases derived from synthetic oligonucleotides) and long (125–147 bases) RNA probes, derived from cloned sequences, could be used to obtain quantitative information on specific mRNA species.

The solution hybridization assay was used to compare the levels of insulin-like growth factor-I (IGF-I) and IGF-II mRNAs in various rat and human tissues. In the rat the liver was the main source of IGF-I mRNA (approximately 400 molecules/cell), but significant levels were also found in extrahepatic tissues such as fat and muscle (3–50 molecules/cell). Human liver contained approximately 100-fold less IGF-I mRNA than rat liver. Human fat, muscle and placenta contained levels of IGF-I mRNA (2–8 molecules/cell) similar to those in the liver. Levels of IGF-II mRNA in rat and human tissues were similar, in that the expression was greatest in the placenta (approximately 200 molecules/cell). Species differences were evident, however, since human liver and fat contained significant amounts of IGF-II mRNA (15–20 molecules/cell), while the rat counterparts had almost undetectable levels. Young and old rats were used to examine the influence of age on the expression of IGF-I and GH receptor mRNAs in the liver. Levels of both IGF-I mRNA and GH receptor mRNA were found to decrease with age (2.8-fold and 1.7-fold respectively).

It is concluded that low levels of IGF mRNAs can be detected using the solution hybridization assay and that there are considerable species differences within and between tissues with regard to steady-state levels of IGF-I and IGF-II mRNAs.
Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu 3, following their expression in Escherichia coli as fusion proteins
Authors: King R. , Wells J. R. E. , Krieg P. , Snoswell M. , Brazier J. , Bagley C. J. , Wallace J. C. , Ballard F. J. , Ross M. , Francis G. L.
Published: December 2008

The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.
Characterization of dopamine receptors associated with steroid secretion in frog adrenocortical cells
Authors: Morra M. , Leboulenger F. , Vaudry H.
Published: December 2008

We investigated the type of receptors involved in the mechanism of action of dopamine on corticosteroid secretion from the frog interrenal (adrenal) gland, using the in-vitro perifusion technique. Exposure of dispersed interrenal cells to 50 μm dopamine for 20 min had a biphasic effect on corticosterone and aldosterone secretion, i.e. a transient stimulation followed by an inhibitory phase. Repeated administration of equimolar pulses of dopamine, given at 150-min intervals, resulted in an enhancement of corticosteroid secretion followed by a subsequent blockade of the stimulatory phase of the response. In contrast, the dopamine-evoked inhibition of corticosteroid release did not show any sensitization or desensitization phenomena. Infusion of repeated pulses of the D1 receptor agonist SKF38393 (32 μm) stimulated corticosteroid release and mimicked the sensitization-desensitization phenomenon induced by dopamine. Repeated administration of the D2 receptor agonist LY171555 (50μm) resulted in a reproducible inhibition of corticosterone and aldosterone secretion. These results suggested the presence of two different receptors for dopamine, i.e. D1 and D2, on frog adrenocortical cells, responsible respectively for the stimulatory and inhibitory effects of dopamine on steroid secretion. However, bromocriptine (50 μm) and CV205-502 (50 μm), two other D2 receptor agonists, had no effect on corticosteroid release. In addition, several classical D2 receptor antagonists failed to block the effect of dopamine on steroidogenesis. It was also observed that (−)sulpiride, a specific D2 antagonist, did not alter dopamine-induced inhibition of inositol phosphate formation. On the other hand, dopamine and the selective D1 and D2 agonists SKF38393 and LY171555 did not affect the formation of cyclic AMP by interrenal tissue.

Taken together, these data indicate that dopamine directly regulates corticosteroid secretion from frog adrenocortical cells. The effect of dopamine is not coupled to adenylate cyclase activity but is probably mediated through the phosphoinositide-turnover pathway. The pharmacological characteristics of the receptors involved in the mechanism of action of dopamine clearly differ from those of the D1 and D2 subtypes previously described in mammals.
Arachidonic acid-induced insulin secretion from rat islets of Langerhans
Authors: Band A. M. , Jones P. M. , Howell S. L.
Published: December 2008

There is growing evidence that arachidonic acid (AA) and/or its metabolites may be involved in the control of insulin secretion. We have now investigated the effect of AA on insulin secretion from rat islets, and the possible involvement of protein kinase C (PKC) in this process. Exogenous AA stimulated insulin secretion from intact islets at a substimulatory concentration of glucose (2 mm), but did not further enhance glucose-induced (20 mm) insulin secretion. AA-induced insulin secretion was temperature dependent. The secretory responses seen at 37°C were totally abolished by reducing the incubation temperature to ≤34°C. AA-induced insulin secretion was not dependent upon extracellular Ca2+ and was potentiated by omission of Ca2+ or bovine serum albumin from the media. PKC in rat islets can thus be stimulated by AA, but the stimulation of PKC is not required for AA-induced insulin secretion.