bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of steroidogenesis by the calcium messenger system in human adrenocortical cells
Authors: Laird S. M. , Hinson J. P. , Vinson G. P. , Mallick N. , Kapas S. , Teja R.
Published: December 2008

The involvement of the calcium messenger system in the control of steroidogenesis in the rat and bovine adrenal cortex has been studied extensively. However the role of these second messengers in the control of human adrenocortical function is not established. This was therefore studied by incubating collagenase-dispersed human adrenocortical cells with the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). The effects of the calcium channel blocker verapamil on basal and stimulated steroidogenesis were also studied.

Both TPA (1 pmol/l–10 μmol/l) and A23187 (1 nmol/l–10 μmol/l) caused a dose-dependent increase in cortisol, aldosterone and corticosterone production. Verapamil (10 μmol/l) inhibited the increase in aldosterone, corticosterone and cortisol produced in response to ACTH(1–24), potassium, and desacetyl-αMSH. Unlike previous results in the rat, these effects were not specific for aldosterone secretion.

The results suggest that, as in other species, calcium mobilization and protein kinase C activation have a role in the control of steroidogenesis in the human adrenal cortex. However, in contrast to the rat, these mechanisms appear to be involved in the control of steroidogenesis in both the zona glomerulosa and inner zone cells.
Cloning and characterization of a gene encoding pig epidermal growth factor
Authors: Pascall J. C. , Jones D. S. C. , Doel S. M. , Clements J. M. , Hunter M. , Fallon T. , Edwards M. , Brown K. D.
Published: December 2008

A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron—exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5′ end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5′ end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.
Rat adrenal 5α-reductase mRNA content and enzyme activity are sex hormone dependent
Authors: Lephart E. D. , Simpson E. R. , Trzeciak W. H.
Published: December 2008

To investigate the effects of sex hormones on 5α-reductase, we examined 5α-reductase mRNA content and enzyme activity in the adrenal cortex of peripubertal male and female rats. In male rats, the influence of castration or hormone-replacement treatment with dihydrotestosterone (5α-DHT) on 5α-reductase was assessed. To stimulate ovarian sex hormone production in immature female rats, the effect of a single injection of 5 IU pregnant mare serum gonadotrophin (PMSG) on 5α-reductase was examined. The efficacy of the treatments was demonstrated by measuring serum LH and ventral prostate weight in male rats, and serum oestradiol and ovarian weight in female rats. Growth hormone was also measured across all treatments in male and female rats. Adrenal 5α-reductase mRNA levels were determined by RNA blot analysis utilizing a rat 5α-reductase cDNA as probe. 5α-Reductase enzyme activity was estimated by isolating [ 3H]5α-DHT by thin-layer chromatography after incubation with [3H]testosterone. The identity of the [3H]5α-DHT formed was demonstrated by recrystallization of the derivatized DHT to constant specific activity. In controls, adrenal cortical 5α-reductase mRNA content was nearly four times higher in immature female rats compared with intact peripubertal males. Castration resulted in a sevenfold increase in adrenal 5α-reductase mRNA content compared with that in intact controls, while in DHT-injected castrated animals the mRNA level was nearly undetectable. The content of adrenal 5α-reductase mRNA in anoestrous rats was nearly four times higher than in PMSG-treated animals. Adrenal 5α-reductase activity was higher in immature female rats than in intact peripubertal males. Castrated rats displayed more than a threefold increase in 5α-reductase activity over that of controls, whereas the activity values were below controls in castrated animals treated with DHT. In immature female rats treated with PMSG, 5α-reductase activity decreased by 40% to that of anoestrous controls. These results indicate that in the rat adrenal cortex the content of mRNA encoding 5α-reductase is negatively regulated by sex hormones presumably at the transcriptional level. Suppression of the enzymatic activity of adrenal 5α-reductase by sex hormones is due to lower mRNA levels encoding this protein.
Expression of parathyroid hormone-related protein mRNA in the rat before birth: demonstration by hybridization histochemistry
Authors: Senior P. V. , Heath D. A. , Beck F.
Published: December 2008

The ontogeny of parathyroid hormone (PTH) and PTH-related protein (PTHrP) gene expression was studied by hybridization histochemistry in the rat at various stages between implantation and full term. PTHrP mRNA was demonstrable in the early post-implantation trophoblastic giant cells but disappeared from this site before 13·5 days. Localized gene expression, detectable by the in-situ technique, began between 12·5 and 15·5 days in embryonic tissues. The distribution of gene expression suggests that PTHrP may be concerned with the process of implantation. Its widespread, yet clearly localized, distribution in embryonic and fetal tissues is consistent with a paracrine or autocrine function which may relate to the transforming growth factor-β family of growth factors. PTH expression occurred solely in the parathyroid and was detectable in the fetal parathyroid at 13·5 days of gestation.
Multiple forms of angiotensin II receptors in rat tissues
Authors: Jimenez E. , Marsigliante S. , Barker S. , Hinson J. P. , Vinson G. P.
Published: December 2008

Angiotensin II (AII) receptors were identified in rat tissue membranes by specific binding of 125I-labelled AII. Using an isoelectric focusing technique, two forms of the high-affinity AII receptor were identified in rat adrenal zona glomerulosa and liver membranes. These migrated to isoelectric points (pI) 6.8 and 6.7. Two low-affinity forms migrated to pI 6.5 and 6.3. The two high-affinity forms were in greatest abundance in the zona glomerulosa, while the low-affinity pI 6.5 isoform was predominant in liver membranes. In uterine membranes both low-affinity isoforms were observed, but there was only one of the high-affinity forms (pI 6.7).

Concentrations of AII receptor isoforms were increased in the zona glomerulosa of sodium-deprived rats.

Reduction of disulphide bridges with dithiothreitol (DTT) had different effects on the various AII receptor isoforms. Thus 1 mmol DTT/l caused a twofold increase in 125I-labelled AII binding in zona glomerulosa membranes. DTT produced no appreciable differences in specific AII binding in uterine membranes, whereas there was a 50% reduction of binding in liver membranes. At 20mmol/1, DTT greatly decreased AII binding in all tissues.

The data suggest the existence of multiple forms of AII receptors which may have different functions.
The gene for the common α subunit of porcine pituitary glycoprotein hormone
Authors: Kato Y. , Ezashi T. , Hirai T. , Kato T.
Published: December 2008

The gene for the common α subunit of the porcine anterior pituitary glycoprotein hormones was cloned from a genomic library constructed in EMBL3. The nucleotide sequence of the entire coding sequence of the porcine common α-subunit gene was determined in addition to one intron and 1059 and 160 bp of the 5′-and 3′-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the common α-subunit gene is present as a single copy. The transcriptional unit of the porcine common α subunit spanned about 14kb and contained four exons interrupted by three introns of about 11.5, 1.2 and 0.4kb. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues +9/+10 and +71/+72 were highly conserved among the rat, human and bovine common α-subunit genes. In the proximal portion of the 5′-flanking region, one TATA box and one CCAAT box were present. A steroid-responsive element was not found up to 1059 bases upstream from the transcription start site. The potential AP-1 and AP-2 factor-responsive elements were present at three and one positions respectively in the 5′-flanking region. This feature suggests that hypothalamic gonadotrophin-releasing hormone stimulates the expression of the common α-subunit gene predominantly by a signal-transduction system, with the protein kinase C cascade and factors AP-1 and AP-2 as mediators. The cyclic AMP-responsive element was also present at two positions, but a single base substitution was found in each sequence compared with the consensus sequence. The porcine common α-subunit gene has a structure distinct from its counterparts, the porcine FSH-β and LH-β genes, reflecting differential control of their synthesis during gametogenesis.
Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion
Authors: McNeilly J. R. , Brown P. , Clark A. J. , McNeilly A. S.
Published: December 2008

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free α subunit.

Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-β mRNA and FSH-β mRNA levels (to 5 and 30% of luteal control values respectively), but not α mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in α mRNA, there was a decrease in free α subunit in both the pituitary and plasma (to 30 and 80% of control levels).

These results suggest that, while GnRH positively regulates the expression of both gonadotrophin β-subunit genes, it can, under certain circumstances, negatively regulate α-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of β-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway. The dramatic reduction in LH pituitary content indicates that GnRH is required for the operation of a regulatory/storage pathway for the secretion of LH. There appears to be no similar mechanism for FSH. The LH-specific pathway is probably dependent upon the availability of LH-β subunits which subsequently plays a role in regulating α subunit by sequestering, assembling and storing the intact hormone in the presence of GnRH. Finally, in the absence of responsiveness to GnRH, the regulation of α-subunit production is not at the level of gene transcription. Inefficient translation of the mRNA or rapid degradation of the free α chain may account for the observed dramatic decrease in production of α subunit.
Modulation of the functional properties of human thyrocytes in monolayer or follicle culture: effects of some anaesthetic drugs
Authors: Massart C. , Le Tellier C. , Mallédant Y. , Leclech G. , Nicol M.
Published: December 2008

It is well known that some volatile anaesthetic drugs, such as halothane and isoflurane, alter the functions of the human thyroid gland, but the action of other anaesthetic drugs, such as thiopental, midazolam and ketamine, on thyroid function is still unknown. We have investigated the effects of these three drugs on the functional properties of human thyrocytes cultured in monolayers or follicles and stimulated by TSH. Thiopental, midazolam and ketamine induced total suppression or a partial reduction, depending on the dose administered, of cyclic AMP (cAMP), follicular thyroglobulin (Tg) and free tri-iodothyronine (FT3) production. In contrast, free thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drugs did not affect thyrocyte production. The inhibiting effect of thiopental, midazolam and ketamine on Tg and FT3 production seems to result from the inhibition of cAMP production and 5′-deiodinase.
Involvement of calmodulin in depolarization-induced release of corticotrophin-releasing hormone-41 from the rat hypothalamus in vitro
Authors: Tsagarakis S. , Rees L. H. , Besser G. M. , Grossman A.
Published: December 2008

We have employed an acute explant system of the rat hypothalamus in vitro, as previously described, to examine the role of calcium and calmodulin in the release of corticotrophin-releasing hormone-41 (CRH-41). Release of CRH-41, as determined by radioimmunoassay, was stimulated in a dose-dependent manner by the membrane-depolarizing agents KCl and veratridine. Stimulation was also observed with the calcium ionophore A23187. The calcium channel blocker verapamil (1–100 μmol/l) inhibited both KCl-and veratridine-induced release in a dose-dependent manner (maximum inhibition of 75% and 60% respectively), thus providing further evidence that calcium entry is required for secretion of CRH-41 following membrane depolarization. Trifluoperazine (1–100 μmol/1), an inhibitor of calmodulin—calcium interaction, decreased both KCl- and veratridine-evoked CRH-41 secretion in a dose-dependent fashion (maximum inhibition of 50% and 30% respectively). Similarly, phenytoin, a calmodulin-dependent kinase inhibitor, in the concentration range of 1–100 μmol/1, also decreased depolarization-induced CRH-41 release in a dose-dependent manner. The basal release of CRH-41 was unaffected by either treatment. Finally, both calmodulin inhibitors (10 μmol/l) decreased CRH-41 release induced by the calcium ionophore A23187 (10 μmol/l).

These data provide evidence for the role of calcium in membrane depolarization-induced stimulus-secretion coupling of rat hypothalamic CRH-41. Furthermore, inhibition of the stimulatory responses by two separate classes of calmodulin inhibitors suggests a role for calmodulin, at least in part, in this process.
TRI-IODOTHYRONINE INCREASES INTRA-CELLULAR CALCIUM LEVELS IN SINGLE RAT MYOCYTES
Authors: Lomax R.B. , Cobbold P.H. , Allshire A.P. , Cuthbertson K.S.R. , Robertson W.R.
Published: December 2008

We have studied the effects of acute administration of tri-iodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to <1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80mM KC1, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150±56nM (mean ± SD, n=14) by 127±47nM on depolarization with 80mM KC1. Application of T3 (1-100nM) led to an increase (P<0.05) in [Ca2+]i (mean amplitude of 152±35nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when lOOnM T3 was applied (median and range; 6.8, 0-14 min) than when 1nM T3 was used (16, 7.0-56.1 min) (P<0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.
Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human proCRH gene: intracellular storage and regulated secretion
Authors: Castro M. G. , Brooke J. , Bullman A. , Hannah M. , Glynn B. P. , Lowry P. J.
Published: December 2008

The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1–76), ACTH, β-lipotrophin and β-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. ArT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1–41). On stimulation with the secretagogue noradrenaline, CRH(1–41) was released into the medium, while the precursor was not. Whilst treatment of wild-type ArT-20 cells with exogenous CRH(1–41) (1 nm) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.
Intracellular signalling in the ovine pars tuberalis: an investigation using aluminium fluoride and melatonin
Authors: Morgan P. J. , Hastings M. H. , Thompson M. , Barrett P. , Lawson W. , Davidson G.
Published: December 2008

The effect of aluminium fluoride (AlF4−) has been studied on inositol phosphate accumulation, calcium mobilization, cyclic AMP production and [2-125I]iodomelatonin binding in ovine pars tuberalis cells. These cells have high-affinity receptors for, and respond to, melatonin through inhibition of forskolin-stimulated adenylate cyclase. In the presence of 10 mm LiCl, AlF4− stimulated the net accumulation of inositol monophosphate and inositol bisphosphate. Consistent with these findings, AlF4− increased intracellular calcium; although this response was attenuated in calcium-depleted medium, indicating that the calcium response comprises both intracellular and extracellular components. Melatonin was ineffective on either basal or AlF4−-stimulated turnover of inositol phosphates. In concordance with the inositol phosphate response, melatonin had no effect on either the AlF4−-stimulated or the basal calcium levels. AlF4− blocked the increase in cyclic AMP stimulation by l μm forskolin, being as effective as melatonin, achieving approximately 90% inhibition. AlF4− also attenuated the binding of [2-125I]iodomelatonin to ovine pars tuberalis membranes by 15%. At the concentration used, these results are consistent with the interpretation that AlF4− activates many G protein-mediated responses, and thus imply that the inhibitory pathway for cyclic AMP predominates over the stimulatory arm, whereas there can only be a stimulatory pathway linked to phosphoinositide metabolism in ovine pars tuberalis cells.
Isolation of the mouse cytochrome P450J (CYP2E1) cDNA and its reciprocal testosterone regulation in kidney and liver
Authors: Voliva C. F. , Paigen K.
Published: December 2008

A hybridization probe that is homologous to the B2 short interspersed repetitive element detects an mRNA in mouse kidney and liver that is regulated by androgen. Administration of testosterone induces this mRNA in kidney and represses it in liver. The mRNA was cloned by first using the B2 probe to select 48 cDNA clones from an androgen-induced kidney library. These clones were then tested for their androgen response by hybridizing them with probes made by reverse transcription of basal and testosterone-treated kidney poly(A)+ RNA. Any homology to the B2 sequence was masked by prehybridizing the filters to an excess of non-radioactive RNA synthesized from a B2 sequence cloned into a riboprobe vector. A unique sequence was subcloned from the largest androgen-responsive cDNA clone. A radioactive riboprobe generated from the unique sequence subclone detected an androgen-responsive mRNA in Northern blots with the same electrophoretic mobility as the predominant androgen-responsive mRNA detected with the B2 homologous riboprobe. The riboprobe also detected a unique sequence in Southern blots of genomic DNA. This subclone was then used as the probe to isolate a full-length cDNA clone from a second androgen-induced kidney library.

When sequenced, this full-length cDNA of an androgen-responsive, B2-containing mRNA showed strong homology to the rat and human cytochrome P450J and the rabbit cytochrome P450 3a genes (CYP2E1). It showed only weak homology to the mouse testosterone 15 α-hydroxylase gene (CYP2A3) which is also regulated reciprocally by androgen in kidney and liver. The sequence of mouse P450J is identical to the B2 homologous mRNA previously named B2+mRNAx which is abundant in mouse liver.
Localization of the sites of synthesis and action of prolactin by immunocytochemistry and in-situ hybridization within the human utero-placental unit
Authors: Wu W.-X. , Brooks J. , Millar M. R. , Ledger W. L. , Saunders P. T. K. , Glasier A. F. , McNeilly A. S.
Published: December 2008

While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.
H+-Dependent ATPase and K+ channel activities in the rat thyroid cell strain FRTL-5
Authors: Woods D. J. , Soden J. , Bidey S. P.
Published: December 2008

Using the fluorescent indicators 2′,7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein and Oxonol V to monitor intracellular pH (pHi) and cell membrane potential respectively, we have investigated the involvement of H+-dependent ATPase and H+-dependent K+ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pHi of 6.42. The subsequent recovery of pHi was unimpaired by the absence of extracellular K+, but was reduced in the presence of the Na+ antagonist amiloride (1 mmol/l), recovering by 0.11±0.003 units, compared with 0.27±0.02 units under amiloride-free conditions. In the presence of the H+-dependent ATPase antagonist N,N′-dicyclohexylcarbodiimide (DCC), the pHi recovery observed in amiloride-containing, K+-free buffer was abolished.

The recovery of pHi in Na+- and K+-containing buffer was accompanied by hyperpolarization of the cell membrane, the later stage of which was reduced after blockade of K+ channels with BaCl2, implying a major contribution of transmembrane K+ movement to such events. In contrast to its attenuating effect on pHi recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H+-dependent ATPase may not be a major contributory factor to this event. However, when K+ channels were blocked by addition of BaCl2, addition of DCC abolished the butyrate-induced membrane depolarization. These findings are consistent with the presence of two independent hyperpolarizing transport processes in the FRTL-5 cell membrane which appear to involve (i) a H+-dependent ATPase, activated in response to cytosolic acidification, and allowing partial recovery of pHi in the absence of extracellular Na+ and HCO3−, and (ii) H+-dependent K+ channels which, while contributing to membrane hyperpolarization, may not play a major role in the normal maintenance of pHi.
A single amino acid residue replacement in the β subunit of human chorionic gonadotrophin results in the loss of biological activity
Authors: Chen F. , Puett D.
Published: December 2008

The heterodimer, human chorionic gonadotrophin (hCG), contains an a subunit that is common to the glycoprotein hormones and a hormone-specific β subunit. A comparison of all known β amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-β) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-β Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine a subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-β cDNAs. The Arg99 β mutant associated with the a subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.
Evidence for two distinct hormone-sensitive [3H]phosphoinositide pools in bovine adrenocortical zona fasciculata/reticularis cells stimulated with angiotensin II
Authors: Bird I. M. , Williams B. C. , Walker S. W.
Published: December 2008

Bovine adrenocortical cells from the zona fasciculata/reticularis were isolated and their phosphoinositides labelled to a steady state with [3H]inositol in primary culture. Experiments performed on these cells in the presence of Li+ have shown that, over a period of 60min, angiotensin II (AII; 10−7m) stimulated a linear increase in [3H] inositol phosphates that was sustained through the utilization of two hormone-sensitive subpools of prelabelled lipid (30% and 45% respectively), and a rapid resynthesis of [3H]phosphoinositide into one of these pools using cytosolic [3H]inositol. The 30% pool was used immediately on stimulation, and was sustained at a steady-state size of 10–15% during the first 30 min of stimulation through rapid resynthesis using cytosolic [3H]inositol. Only after 30min, when the cytosolic [3H]inositol was depleted and resynthesis could no longer occur, did the additional 45% pool start to supply further substrate to the phospholipase C, thereby further sustaining the generation of [3H]inositol phosphates. Once this pool was depleted however (by approximately 60min), [3H]inositol phosphate generation finally ceased. These findings establish the differential use of two metabolically distinct hormone-sensitive pools of phosphoinositide following AII stimulation in bovine adrenocortical cells, events which are dependent upon the availability of cytosolic inositol for phosphoinositide resynthesis.
Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells
Authors: Broadhead C. L. , O'Sullivan U. T. , Deacon C. F. , Henderson I. W.
Published: December 2008

The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in freshwater (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles.

Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site.

Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells.

These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.
Effect of somatostatin on prolactin in rainbow trout (Oncorhynchus mykiss) pituitary cells in primary culture
Authors: Goff P. Le , Weil C. , Valotaire Y. , Gonnard J. F. , Prunet P.
Published: December 2008

To study the control of prolactin secretion in fish, an in-vitro technique using a monolayer cell culture system of rainbow trout pituitary glands was developed. Such secretion was characterized by measurement of both prolactin release and prolactin mRNA content using a trout prolactin cDNA as a probe. This cell culture technique, already used to study the regulation of gonadotrophin secretion in rainbow trout, was further validated by measuring total DNA and protein content. Both parameters appeared to be stable after 2 days of culture. Studying the effect of somatostatin (SRIF) on prolactin cells indicated that a maximal inhibitory effect (62%) was observed after 24 h of treatment. Significant inhibition of prolactin release was obtained for SRIF doses ranging from 50 nm to 1 μm. However, in the same experiment, SRIF was much more potent as an inhibitor of growth hormone release. Short-term (<12h) incubation with SRIF did not induce a significant change in prolactin release, whereas growth hormone release was reduced at as early as 1 h after SRIF exposure. SRIF did not have a significant effect on total prolactin content or prolactin mRNA levels, suggesting the absence of an effect on prolactin synthesis. No increase in the magnitude of the inhibitory effect of SRIF was observed when using pituitary cells from immature, mature male or mature female trout. When comparing effects on primary cultures containing cells from the whole pituitary with a prolactin cell-enriched population, SRIF appeared to have the same inhibitory effect on prolactin release, supporting a direct action of SRIF on prolactin cells. These results provide further support for SRIF being a prolactin-inhibiting factor in rainbow trout and acting as a modulator of a dominant stimulatory control of prolactin release.
Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry
Authors: Clayton R. N. , Eccleston L. , Gossard F. , Thalbard J.-C. , Morel G.
Published: December 2008

There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used insitu hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary→Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.
Transforming growth factor-β1 production in porcine thyroid follicular cells: regulation by intrathyroidal organic iodine
Authors: Cowin A. J. , Davis J. R. E. , Bidey S. P.
Published: December 2008

The present studies have demonstrated the production of transforming growth factor-β1 (TGF-β1) by porcine thyroid follicular cells (TFCs) maintained in vitro as subconfluent monolayers, and have confirmed a stimulatory effect of iodide on thyroidal TGF-β1 mRNA and peptide release. RNA extracted from TFCs maintained in the absence of iodide contained a 2·5 kb transcript which hybridized specifically with a cDNA probe for human TGF-β1, and which showed an approximate doubling in intensity in cells exposed to 10 μmol NaI/l. In the presence of the anti-thyroid thionamide drug methimazole (MMI; 1 mmol/l), the action of iodide on TGF-β1 mRNA was attenuated, although MMI alone had no effect on the control level of TGF-β1 mRNA. The TGF-β1 peptide content of TFC-conditioned media (TFC-CM) was assessed using the fetal mink lung cell line Mv1 Lu, in which activated TGF-β1 specifically suppresses trichloroacetic acid-precipitable [methyl-3H]thymidine incorporation. Newly conditioned TFC-CM stimulated [methyl-3H]thymidine incorporation into Mv1Lu cells, but after heat treatment to inactivate growth stimulators and activate the latent TGF-β1 component this medium inhibited [methyl-3H]thymidine incorporation. This inhibitory effect was prevented by immunoadsorption of TFC-CM with a TGF-β1-neutralizing antiserum, confirming the specificity of the inhibitory response. The inhibitory activity of TFC-CM was increased when the TFCs were preincubated with 10 μmol NaI/l, and lost when TFCs were exposed to MMI. In conclusion, TFCs produce TGF-β1 mRNA and TGF-β1 peptide, which are both increased by iodide treatment in vitro. The anti-thyroid effects of MMI may, at least in part, be mediated by a decrease in TFC-derived TGF-β1 production.
Intraperitoneal insulin is more potent than subcutaneous insulin at restoring hepatic insulin-like growth factor-I mRNA levels in the diabetic rat: a functional role for the portal vascular link
Authors: Russell-Jones D. L. , Rattray M. , Wilson V. J. , Jones R. H. , Sönksen P. H. , Thomas C. R.
Published: December 2008

There is evidence that the hormonal control of hepatic IGF-I production is mediated by GH and insulin. To elucidate the role of these hormones further we administered s.c. or i.p. insulin (at 2·5 and 5·0 IU/day) and/or GH (0·8 IU/day) to rats made diabetic with streptozotocin 16 days previously. Hepatic IGF-I production was then assessed by quantifying hepatic IGF-I mRNA levels by autoradiography of Northern blots. Diabetes resulted in a fivefold reduction in hepatic IGF-I mRNA levels (optical density (OD) of the 0·7–1·1 kb band: controls, 1·3±0·09; diabetics, 0·28±0·08; P<0·01), which was not significantly changed by treatment with s.c. insulin (OD: low dose, 0·55±0·05; high dose, 0·58±0·05) or low dose i.p. insulin (OD: 0·40±0·03). High dose i.p. insulin enhanced hepatic IGF-I mRNA levels (OD: 0·93±0·23) compared with diabetic rats (P<0·01) and those given high dose s.c. insulin (P<0·04), despite the blood glucose values being similar in the treated groups (i.p., 4·72±0·29 mmol/l; s.c., 3·32±0·03 mmol/l). Administration of GH alone partially restored the hepatic IGF-I mRNA level (OD: GH-treated, 1·00±0·05; diabetic, 0·28±0·08; P<0·01), whilst having no effect on blood glucose values (diabetic, 36·35±0·45 mmol/l; GH-treated, 38·65±2·39 mmol/l). Additional administration of s.c. insulin completely restored IGF-I mRNA levels to those of controls (OD: low dose, 1·35±0·14; high dose, 1·27 ± 0·18). These observations indicate that insulin and GH are required for full expression of hepatic IGF-I mRNA and that insulin given i.p. is more potent than that given s.c. at stimulating hepatic synthesis of IGF-I.
Use of pertussis toxin to investigate the mechanism of action of prostaglandin F2α on the corpus luteum in sheep
Authors: McCann T J , Flint A P F
Published: December 2008

Pertussis toxin catalysed the ADP-ribosylation of a protein of Mr 40 000 in ovine luteal tissue. Ribosylation of 45% of this protein in whole cell incubations (as judged by subsequent ribosylation of cell-free preparations in the presence of [32P]NAD) attenuated the prostaglandin (PG)F2α-stimulated hydrolysis of [3H]inositol-labelled phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate by 60%, but did not affect the inhibition by PGF2α of LH-stimulated accumulation of cyclic AMP. It is concluded that activation of phospholipase C by PGF2α involves a pertussis toxin-sensitive protein, probably a G protein, and that the inhibitory effect of PGF2α on LH-stimulated adenylate cyclase is unlikely to be directly mediated by such a protein.
Insulin-like growth factor-I gene expression is increased in the right ventricular hypertrophy induced by chronic hypoxia in the rat
Authors: Russell-Jones D L , Leach R M , Ward J P T , Thomas C R
Published: December 2008

Rats were maintained in chambers and breathed air (control, n=8) or an atmosphere containing 10% oxygen (hypoxic, n=10) for 35 days. On completion of the experiment the hypoxic animals weighed less than the controls (hypoxic, 290 ± 11.7g; control, 339 ± 19.2g; means ± S.E.M., p<0.05). No differences in the left ventricular weights were found between groups but the right ventricular weights were greater in the hypoxic rats (hypoxic, 0.39 ± 0.02g; control, 0.27 ± 0.08g; p<0.01). The amount of mRNA for IGF-I in the ventricles was quantified by Northern blot analysis. There was no difference between groups in IGF-I mRNA levels in the left ventricles (hypoxic, 1.07 ± 0.41 absorbance units (AU); control, 0.73 ± 0.33 AU). In the right ventricles, IGF-I mRNA was greater in hypoxic than in control rats (hypoxic, 2.37 ± 0.75 AU; control, 0.64 ± 0.11 AU; p<0.05). This study demonstrates that expression of IGF-I mRNA is increased in the hypertrophied right ventricle of hypoxic rats; IGF-I may play a central role in the initiation and maintenance of this process.
Nitric oxide stimulates cyclic GMP in human thyrocytes
Authors: Millatt L J , Jackson R , Williams B C , Whitley G StJ
Published: December 2008

Sodium nitroprusside spontaneously breaks down in solution to produce the vasodilator nitric oxide. In many cell types, this stimulates the cytosolic form of the enzyme guanylate cyclase, resulting in the elevation of cyclic GMP (cGMP). We have investigated the effect of sodium nitroprusside on the generation of cGMP in primary human thyrocytes and the SV40-transfected human thyroid cell line SGHTL-189. A dose-dependent increase in cGMP was obtained and the maximum response was observed with concentrations above 10 μm sodium nitroprusside in both cell types. Methylene blue (50 μm) had no significant effect on basal cGMP production but inhibited the effect of sodium nitroprusside at all concentrations tested, thus demonstrating that the effect was due to nitric oxide. Sodium nitroprusside had no effect on cyclic AMP (cAMP) production in these cells. TSH at 100 and 1000 μU/ml significantly stimulated the production of cAMP, but not that of cGMP, in primary human thyrocytes. Sodium nitroprusside had no significant effect on basal or TSH-stimulated triiodothyronine secretion in primary human thyrocytes. Forskolin (10 μm) significantly stimulated cAMP production in both primary thyrocytes and SGHTL-189 cells. Although forskolin had no significant effect on basal cGMP production, sodium nitroprusside-stimulated cGMP production was significantly reduced by forskolin. However, this inhibitory effect was not related to the production of cAMP.
Mechanisms of ACTH- and angiotensin II-stimulated 1α-hydroxycorticosterone secretion in the dogfish, Scyliorhinus canicula
Authors: Armour K J , O'Toole L B , Hazon N
Published: December 2008

An isolated perifused interrenal gland preparation from the lesser-spotted dogfish, Scyliorhinus canicula, was used to investigate the mechanisms of action of ACTH and angiotensin II (AII) on elasmobranch adrenocortical cells. ACTH-stimulated 1α-hydroxycorticosterone secretion was unaffected by dantrolene and significantly decreased in the absence of extracellular calcium. Dibutyryl cyclic AMP produced a dose-dependent increase in 1α-hydroxycorticosterone secretion. The results suggest that the mechanism of ACTH action in elasmobranchs may be similar to that reported for mammals and amphibians, involving the synergistic action of calcium with the cyclic AMP messenger system. AII-stimulated 1α-hydroxycorticosterone secretion was significantly inhibited in the presence of dantrolene and in the absence of extracellular calcium, indicating that both extracellular and intracellular calcium are required for the full action of AII. These results are consistent with results in mammals and amphibians where AII stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and changes in intracellular calcium concentration, and they suggest that AII may operate via this mechanism to stimulate 1α-hydroxycorticosterone secretion in elasmobranchs.
Regulation of the growth hormone receptor during liver regeneration in the rat
Authors: Husman B , Andersson G
Published: December 2008

The GH receptor (GHR) in regenerating rat liver following partial hepatectomy has been characterized in terms of mRNA levels, size of receptor variants, ligand binding, endocytosis and degradation. The GHR mRNA level, quantitated by solution hybridization, was found to be unchanged during the first 3 days of regeneration. The specific binding of 125I-labelled bovine GH to a GHR-enriched low density membrane fraction was, in contrast to the mRNA level, reduced to 10–35% between 12 and 24 h after partial hepatectomy, and remained low until 48 h. Removal of endogenous GH by MgCl2 treatment did not further increase the binding of 125I-labelled bovine GH. Affinity cross-linking experiments revealed reduced labelling of a specific receptor binder at 95 kDa, assuming a 1:1 stoichiometric binding of the ligand, in regenerating liver. Additional evidence for a decrease in the amount of functional GHR was provided by the finding of decreased levels of IGF-I mRNA in the liver remnant, with a nadir at 50% between 24 and 48 h after hepatectomy. GH binding was reduced to a similar extent in hypophysectomized animals subjected to partial hepatectomy, implying that pituitary GH is not involved in the mechanism of GHR down-regulation.

The in-vivo uptake of 125I-labelled bovine GH, which is correlated with the number of GHRs available for binding in the plasma membrane, was decreased to 23% in an endosome-enriched membrane fraction from regenerating liver at 24 h. No qualitative differences in the size distribution of endocytosed receptor-ligand complexes in normal and regenerating liver were noted. The degradation of internalized 125I-labelled bovine GH, analysed as trichloroacetic acid solubility, was unaffected during regeneration.

It is concluded that the reduced number of GHRs available for binding in regenerating liver cells is caused by an alteration exerted at a post-translational level during receptor biosynthesis.
Partial characterization of a novel oestrogen-induced protein in the rat adenohypophysis
Authors: Casabiell X , Zugaza J L , Pombo C M , Bokser L , Mulet N , Casanueva F F
Published: December 2008

In order to detect putative markers of prolactin-secreting pituitary tumours, adult rats were subjected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography.

Oestrogen induced the appearance in the incubation medium of a protein (OE2 band) with an Mr of 38 000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The protein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues.

No alteration in the rate of generation or the electrophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incubation with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen-induced protein. Both immunoprecipitation experiments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin.

In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent Mr of 38 000, which has at least one intramolecular disulphide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts.
ISOLATION AND STRUCTURE OF A PARTIAL SHEEP OXYTOCIN RECEPTOR cDNA AND ITS USE AS A PROBE FOR NORTHERN ANALYSIS OF ENDOMETRIAL RNA
Authors: Stewart H J , Stevenson K R , Flint A P F
Published: December 2008

The polymerase chain reaction (PCR) was used to generate a 131 bp cDNA encoding part of the sheep endometrial oxytocin receptor. The nucleotide sequence of this cDNA was 93.8% identical to the human oxytocin receptor sequence in this region. When used to probe Northern blots of sheep endometrial RNA the PCR product identified a 6.7 kb mRNA which appeared and disappeared during the oestrous cycle in parallel with the oxytocin receptor molecule as measured by ligand binding. The sheep endometrial receptor mRNA was significantly larger than the human myometrial mRNA (4.7 kb). It is suggested that the cloned cDNA described here is an appropriate probe for use where it is required to measure sheep oxytocin receptor mRNA.