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W Ge, M Tanaka, M Yoshikuni, Y Eto and Y Nagahama

ABSTRACT

We have cloned a full length cDNA coding for the activin type IIB receptor (GactRIIB) from the goldfish ovary. GactRIIB shares 73 and 70% amino acid identity in the extracellular domain, and 78 and 80% identity in the intracellular domain with the type IIB receptors of the mouse and Xenopus respectively. The intracellular domain of GactRIIB contains two serine kinase consensus sequences, DFKSRN and GTRRYMAPE, in agreement with the reports in other vertebrates that serine/threonine phosphorylation is involved in activin signal transduction. The identity of GactRIIB was confirmed by transient expression in the COS cells followed by activin binding. Iodinated human activin A bound to the GactRIIB-transfected cells and the binding could be completely inhibited by unlabeled activin. Affinity labeling revealed a band of about 85 kDa, which is in agreement with the reported type II receptors in other vertebrates. Together with the fact that activin is expressed in the goldfish ovary, the cloning of activin receptors from the ovary suggests paracrine and autocrine roles for activin in the goldfish ovarian functions.

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A J Notini, R A Davey, J F McManus, K L Bate and J D Zajac

( Brinkmann et al. 1999 ). We define this action of the AR as genomic. Androgens can also rapidly induce second messengers, such as kinase signalling cascades. Some effects of androgens have been observed in the presence of inhibitors of transcription and

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M Tena-Sempere, L Pinilla and E Aguilar

ABSTRACT

In the present work in vitro GH pituitary responsiveness to GHRH in short-term (STO) and long-term orchidectomized (LTO) male rats was compared. In agreement with previous data obtained in vivo, pituitaries from STO rats showed reduced GH release after GHRH stimulation while LTO male pituitaries presented responses similar to those from control animals after maximal GHRH (10-6 m) stimulation. This suggests that compensatory mechanisms have taken place, probably at the pituitary level, in order to restore GH pituitary responsiveness to high doses of GHRH. However, LTO male rats showed a reduced sensitivity to GHRH relative to intact males, as indicated by a higher EC50 vs controls (40·82 ± 12·03 nm vs 0·35 ± 0·09 nm in intact males). We aimed to investigate further the events involved in the compensatory mechanisms that take place in LTO rats. For this purpose, we compared in vitro GH secretion by pituitaries from intact and LTO male rats after stimulation with specific activators of the signal transduction pathways related to GH release. Forskolin and dibutyryl cyclic-adenosine 3′,5′-monophosphate were more effective in eliciting GH secretion (expressed in terms of percent increment over basal GH release) in LTO males, whereas phorbol 12-myristate 13-acetate was completely ineffective in stimulating GH release in this group. Thus, our results clearly showed that long-term orchidectomy enhances the effectiveness of the cAMP pathway in inducing GH release while it completely blunts that of the protein kinase C pathway. In conclusion, orchidectomy decreased the effectiveness of GHRH in eliciting GH release in vitro. However, long-term orchidectomy activated compensatory mechanisms that restored complete GH pituitary responsiveness to maximal GHRH stimulation. These mechanisms seem not to operate in STO rats. An increased effectiveness of the cAMP pathway in eliciting GH release in LTO rats is probably involved in the aforementioned compensatory mechanisms.

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K. V. Patel and M. P. Schrey

ABSTRACT

Inositol lipid hydrolysis was monitored in the human breast cancer cell line MCF-7 in response to various bombesin (BN) and substance P (SP) analogues. Both members of the BN family of peptides, i.e. BN and gastrin-releasing peptide (GRP), stimulated a dose-related increase in total inositol phosphate production, with a similar half-maximal effective dose (ED50) around 1 nm. The BN receptor antagonist [Leu13-Ψ-CH2NH-Leu14]-BN (LLBN) at 1 μm was devoid of agonist activity and displaced the BN dose—response to the right, resulting in a tenfold increase in the ED50 for BN. BN also stimulated a dose-related increase in 45Ca2+ efflux which was also inhibited by LLBN. Two SP analogues [dArg1, d-Pro2, d-Trp7,9,Leu11]-SP and [d-Arg1, d-Phe5, d-Trp7,9,Leu11]-SP ([APheTL]-SP), both antagonized BN-stimulated inositol lipid hydrolysis. [APheTL]-SP (60 and 80 μm) alone also exhibited considerable agonist activity which was not antagonized by LLBN. Indeed, a sub-threshold dose of [APheTL]-SP (40 μm) in the presence of LLBN (10 μm) potentiated the inositol lipid hydrolysis response. BN, GRP, LLBN and [APheTL]-SP all inhibited binding of 125I-labelled GRP to MCF-7 cells, to 50% of that occurring in the absence of the peptides, at concentrations of 150 pm, 150 pm, 150 nm and 600 nm respectively. These data are consistent with the presence of separate but interacting receptors or binding sites for BN and SP analogues, which are coupled to a common signal transduction pathway in human breast cancer cells.

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T. Ezashi, T. Hirai, T. Kato, K. Wakabayashi and Y. Kato

ABSTRACT

The gene for the β subunit of porcine LH (LH-β) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-β in addition to 1277 and 372 bp of the 5′- and 3′-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-β gene is present as a single copy. The transcriptional unit of porcine LH-β spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues −16/−15 and +41/+42 were highly conserved in the rat, human and bovine LH-β genes. In the 5′-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5′-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-β gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-β gene was the presence of clusters of GC boxes and CACCC elements in the 5′-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-β gene shows a structure distinct from the porcine FSH-β and common α genes, which are counterparts of the LH-β gene, reflecting differential control of their synthesis during gametogenesis.

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Y. Kato, T. Ezashi, T. Hirai and T. Kato

ABSTRACT

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.

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B. Dattatreyamurty, R. A. Smith, S.-B. Zhang, T. A. Santa-Coloma and L. E. Reichert Jr

ABSTRACT

A 240 kDa protein isolated from bovine calf testis has been shown to have properties characteristic of an FSH receptor. However, rat testis FSH receptor has, on the basis of cloning experiments, been found to have a much lower molecular mass of 75 kDa (peptide only). To examine this point, the size of the FSH receptor in membranes obtained from cultured Sertoli cells of immature rats was determined after polyacrylamide gel electrophoresis under non-reducing conditions, followed by transfer to polyvinylidene difluoride membranes and direct identification of the FSH receptor by ligand blot analysis utilizing radioiodinated human FSH. In this system, the rat Sertoli cell membrane FSH receptor also showed a molecular mass of 240 kDa. Bovine testis contains LH and FSH receptors. We compared the sizes of FSH and LH receptors present in the same bovine testis membrane preparation by ligand blot analysis. The FSH receptor again showed a molecular mass of 240 kDa, whereas the LH receptor showed a molecular mass of 90 kDa. The latter value is similar to that deduced by cloning techniques (75 kDa, peptide only). The evidence seems to suggest that, whereas the molecular mass deduced for the LH receptor on the basis of its cDNA is similar to that of the mature membrane receptor, the size of the FSH membrane receptor is considerably different from that deduced on the basis of its cDNA, presumably as a result of post-translational processing. The marked difference in size between mature FSH (240 kDa) and LH (90 kDa) receptors may reflect significant structural differences of importance with regard to mechanisms of signal transduction.

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C Massart, J Gibassier, N Genetet, M L Raoul, M Baron, F Le Gall and C Lucas

ABSTRACT

We studied the lymphocyte-induced alterations in hormonal metabolism and the production of tumour necrosis factor α (TNF-α) during coculture of thyrocytes and autologous lymphocytes from 20 patients with Graves' disease and from five normal subjects. Thyroglobulin (Tg) mRNA was assessed by slot-blot analysis under TSH stimulation. Tg, tri-iodothyronine (T3) and cAMP secretion in the presence of TSH were measured by RIA after 3 or 5 days of coculture. TNF-α levels produced after 5 days incubation were also assayed in lymphocyte culture and coculture media.

Lymphocytes isolated from peripheral blood (PBLs) altered the production of Tg, T3 and cAMP in autologous thyrocytes. Intrathyroidal lymphocytes (ITLs) decreased Tg and cAMP secretion but had no effect on T3 secretion. The reductions in Tg and cAMP levels obtained with mechanically isolated ITLs (M-ITLs) were generally higher than those obtained with ITLs isolated by dispase (D-ITLs). No difference was seen between Graves' disease and normal cocultures. PBLs secreted large concentrations of TNF-α, larger than those obtained with M-ITLs whereas D-ITLs produced low amounts of this cytokine. In coculture, TNF-α levels were lower than those observed in lymphocyte culture. Significant correlations were obtained between TNF-α levels and the decrease in Tg, T3 and cAMP concentrations. The percentage of T lymphocytes was higher in PBLs and D-ITLs than in M-ITLs. B lymphocytes levels were higher in ITLs, especially M-ITLs, than in PBLs. TNF-α production by B lymphocytes was maximal in M-ITLs.

In conclusion, lymphocytes induced a decrease in hormonal thyroid metabolism when cocultured with autologous thyrocytes. These perturbations may be attributed, at least partly, to TNF-α secreted by lymphocytes. TNF-α interacts via the adenylate cyclase pathway of TSH signal transduction.

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D Ratovondrahona, M Fahmi, B Fournier, MF Odessa, R Skryma, N Prevarskaya, J Djiane and B Dufy

There is still only limited understanding of the early steps of prolactin (PRL) signal transduction in target cells. Recent studies have identified some of the essential first steps: these include the rapid association of the PRL receptor with JAK tyrosine kinases and tyrosine phosphorylation of a number of proteins, including members of the signal transducer and activator of transcription (Stats) family. On the other hand, binding of PRL to its receptor is rapidly followed by calcium influx. However, PRL-induced ionic events and the related ionic channels involved have not been clearly established. This work was undertaken to characterise the channels responsible for calcium influx and to obtain an insight into their activation processes. Using the patch-clamp technique in the cell-attached configuration, single Ca2+ channel currents were recorded following PRL application (10 nM) in Chinese hamster ovary (CHO) cells stably expressing PRL receptor (CHO-E32). Statistical analysis showed that the recorded currents were voltage-independent, with a slope conductance of 16 pS. Although these channels were present in excised patches, the fact that PRL was unable to activate them suggested that a soluble cytoplasmic component may be required. Application of the purified inositol phosphate, Ins(1,3,4,5)P4 (2 microM), to the inside of the excised patch membrane activated the voltage-independent 16 pS Ca2+ channel. The open probability (Popen) was enhanced. The inositol phosphates Ins(1,2,3,4,5)P5 and Ins(1,4,5)P3 did not affect channel activity while InsP6 (20 microM) had some effect, although less marked than that of Ins(1,3,4,5)P4. Using the anion-exchange HPLC technique, we then studied the effects of PRL (10 nM) on the turnover of inositol phosphates (InsPs) in CHO-E32. Our studies showed that PRL induces rapid increases in the production of Ins(1,3,4,5)P4 (207% at 30 s), InsP5 (171% at 30 s), and InsP6 (241% at 30 s). Conversely, Ins(1,4,5)P3 showed a transient decrease at 5 s, accompanied by a concomitant increase in Ins(1,3,4,5)P4, suggesting that the former could be transiently phosphorylated to produce the latter. Comparison of the production kinetics of Ins(1,4,5)P3, Ins(1,3,4,5)P4, InsP5, and InsP6 indicated the possibility of additional metabolic routes which have yet to be determined. This study suggests that PRL promotes Ca2+ entry through voltage-independent Ca2+ channels that may be activated by Ins(1,3,4,5)P4 and InsP6.

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B Lee, PG Bradford and SG Laychock

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP3R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP3R mRNA to beta-actin mRNA were 0.08 +/- 0.02, 0.08 +/- 0.03 and 0.25 +/- 0.04 respectively. Types I, II and III InsP3R mRNA were also expressed in rat (RINm5F) and mouse (betaHC9) pancreatic beta-cell lines, and rat cerebellum. Type III InsP3R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In betaHC9 cells, types II and III InsP3R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP3R mRNA in cerebellum. Culture of betaHC9 cells for 5 days at 2.8 and 25 mM glucose, or RINm5F cells for 7 days at 5.5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP3R mRNA in the cells at the higher glucose concentrations. During short-term (0.5-2 h) incubations, betaHC9 cell type III InsP3R mRNA levels increased in response to glucose in a time- and concentration-dependent manner. Actinomycin D inhibited the glucose response. Alpha-ketoisocaproic acid also stimulated betaHC9 cell type III InsP3R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3-O-methylglucose were without effect. The different levels of expression of mRNA for three InsP3R isoforms in islets and insulinoma cells, and the influence of glucose and alpha-ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP3R subtypes may be unique with each playing a distinct role in beta-cell signal transduction and insulin secretion.