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ABSTRACT

Insulin-like growth factor-II (IGF-II) is a polypeptide mitogen which is believed to play an important role in fetal development. The human and rat IGF-II genes are complex transcription units, which contain multiple promoters and polyadenylation sites and which exhibit alternate splicing of their primary transcripts. In order to study IGF-II gene expression during chick embryonic development, we screened a 10-day chick embryo cDNA library with a human IGF-II cDNA probe. We isolated a clone, designated cigf, that was comprised, in part, of sequences homologous to the second coding exon of the human, mouse and rat IGF-II genes. Comparison of the nucleotide sequence of cigf with that of the corresponding genomic clone indicated that cigf was derived from a spliced antisense transcript of the chicken IGF-II gene, which overlapped the second coding exon. Northern blotting experiments with single-stranded RNA probes synthesized using cigfDNA as a template showed that stage 22 and stage 36 chick embryos contained sense strand IGF-II transcripts of 1.4, 2.2, 4.7 and 7.0kb and antisense strand transcripts of 0.7, 1.3, 1.8, 2.5, 4.9, 6.0 and 8.0kb. The pattern of sense strand IGF-II transcripts was similar to that previously found in rat fetal tissues. Whilst there are precedents for the transcription of both strands of a single gene, this is the first evidence for antisense transcription of an IGF gene. The functions of the antisense transcripts remain to be determined. These findings demonstrate a further level of complexity in the IGF-II transcription unit and indicate that studies of IGF-II transcript distribution performed with double-stranded probes should be interpreted with caution. They also suggest explanations for the recent finding that IGF-II peptides are present at much lower levels in embryos than expected from the high levels of IGF-II transcripts present.

ABSTRACT

pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO₃− ions, FRTL-5 cells regulate intracellular pH (pH_i) by an Na⁺/H⁺ antiport but HCO₃−-dependent exchangers cannot operate. We have investigated pH_i regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO₃− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na⁺/H⁺ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO₃ −-dependent mechanisms), ionic substitution and by NH₄ ⁺/NH₃ (10mm) acid loading. Basal pH_i was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO₃ ions. In HCO₃ −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pH_i of 7·35±0·06. This recovery was Na⁺-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO₃ ⁻-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na⁺ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na⁺ from HCO₃ ⁻-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl⁻ alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na⁺ and presence of amiloride, alkalinization was still seen on the removal of Cl⁻, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pH_i by two Na⁺-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na⁺-independent, Cl⁻/HCO₃− mechanism.

ABSTRACT

Thyroid hormones are essential for the normal growth and development of many tissues. In the rat, hypothyroidism is associated with growth impairment, and hyperthyroidism with the development of a hypercatabolic state and skeletal muscle wasting but, paradoxically, cardiac hypertrophy. The mechanism by which thyroid hormone produces cardiac hypertrophy and myosin isoenzyme changes remains unclear. The role of IGF-I, an anabolic hormone with both paracrine and endocrine actions, in producing cardiac hypertrophy was investigated during this study in hyperthyroid, hypothyroid and control rats. A treated hypothyroid group was also included in order to assess the effect of acute normalization of thyroid function.

Body weight was significantly lower in the hyperthyroid (mean±s.e.m.; 535·5±24·9 g, P<0·05), hypothyroid (245·3±9·8 g, P<0·001) and treated hypothyroid (265·3±9·8 g, P<0·001) animals when compared with controls (618·5±28·6 g). Heart weight/body weight ratios were, however, significantly increased in the hyperthyroid (2·74 ± 0·11×10⁻³, P<0·01) and treated hypothyroid (2·87±0·07 ×10⁻³, P<0·001) animals when compared with controls (2·26±0·03 × 10⁻³). Serum IGF-I concentrations were similar in the control and hyperthyroid rats (0·91±0·07 vs 0·78±0·04 U/ml, P=0·26), but bioactivity was reduced by 70% in hyperthyroid serum, suggesting a circulating inhibitor of IGF. Serum IGF-I levels (0·12±0·03 U/ml, P<0·001) and bioactivity (0·12±0·04 U/ml, P<0·001) were significantly lower in the hypothyroid group. Liver IGF-I mRNA levels were not statistically different in the control and hyperthyroid animals, but were significantly reduced in the hypothyroid animals (P<0·05 vs control). Heart IGF-I mRNA levels were similar in the control and hypothyroid rats, but were significantly increased in the hyperthyroid and treated hypothyroid animals (increased by 32% in hyperthyroidism, P<0·05; increased by 57% in treated hypothyroidism, P<0·01). Cardiac IGF-I was significantly elevated in hyperthyroidism (0·16±0·01 U/mg heart tissue, P<0·01), was low in hypothyroidism (0·08±0·01 U/mg, P<0·01) and was normalized in the treated hypothyroid group (0·11 ± 0·01 U/mg vs control, 0·13±0·01 U/mg).

Low body mass during both hypothyroidism and hyperthyroidism is therefore associated with reduced systemic IGF bioactivity. In hypothyroidism there is a primary defect in the endocrine function of IGF-I, while in hyperthyroidism serum IGF bioactivity is reduced in the presence of normal endocrine production of this anabolic hormone. In contrast, the paracrine actions of IGF-I are increased in the heart during hyperthyroidism, and this hormone appears to play a part in the development of hyperthyroid cardiac hypertrophy.

ABSTRACT

We have isolated the TRH receptor (TRH-R) from a rat anterior pituitary cDNA library, determined its sequence and examined its functional characteristics. Expression studies were carried out in Xenopus oocytes and in COS-7 cells. Microinjection of sense RNA transcripts into Xenopus oocytes showed electrophysiological responses of between 800 and 1000 nA under voltage-clamp conditions. COS-7 cells were transiently transfected with the cDNA clone under the control of a cytomegalovirus promoter and inositol phosphate (IP) measurements carried out. In TRH-R transfected cells, TRH (100 nm) produced an approximately twofold increase in total IP production. In-situ hybridization in the rat anterior pituitary revealed a heterogeneous distribution of label, a characteristic pattern of TRH-R expression. The rat 3·3 kb insert coded for a protein of 411 amino acids compared with 393 for the mouse protein. Over its length, the rat TRH-R protein showed considerable homology with that of the mouse, except for a deletion of 232 bp in the 3′-coding region. This deletion did not appear to affect the functional characteristics of the receptor, as shown by electrophysiological studies with Xenopus oocytes and by transfection of the cDNA into COS-7 cells. The sequence given for the 3′-untranslated region is 1·5 kb longer than that reported for the mouse receptor, and extends to the poly(A) tail.

ABSTRACT

A monoclonal antibody (BPL-M23) to insulin-like growth factor-I (IGF-I) was obtained following immunization of BALB/c mice with human IGF-I conjugated to ovalbumin. The affinity constant of BPL-M23 for IGF-I was 10·5 litres/nmol and the cross-reactivities of IGF-II, multiplication-stimulating activity III-2 and insulin were 08, 003 and less than 0·0001 % respectively. Porcine, bovine, ovine and rabbit sera, but not rat or mouse sera, showed substantial reactivity with the antibody.

Comparison of radioimmunoassay analyses of 54 human serum samples from normal subjects and acromegalic and GH-deficient patients using BPL-M23 and a polyclonal rabbit antiserum (R557A) to human IGF-I showed a high correlation, indicating the usefulness of the monoclonal antibody in radioimmunoassay.

Monoclonal antibody BPL-M23 was capable of abolishing the sulphation, mitogenic and insulin-like activities of IGF-I in in-vitro bioassays, suggesting that these activities may rely upon the same receptor-binding site which is near to the antibody-binding site.

ABSTRACT

Expression of receptors for the hypothalamic regulatory peptide, gonadotrophin-releasing hormone (GnRH), was investigated by intracellular recording from Xenopus oocytes injected with poly(A)⁺ mRNA isolated from rat anterior pituitary glands. Membrane depolarizations were induced in oocytes in a dose-dependent fashion following the application of GnRH (10nM - 1μM) or a GnRH superactive agonist, buserelin (1nM - 1μM). The response was reversibly blocked by the addition of a GnRH antagonist (1μM). TRH (10nM - 1μM) had no effect on most of these oocytes. In contrast, some other oocytes which showed no responses to GnRH or to the GnRH agonist, displayed depolarizing responses to TRH (10nM - 1μM). A relatively small number of oocytes responded to both ligands. Control oocytes did not respond to the GnRH analogues or to TRH. This successful expression of the GnRH receptor could provide a new approach to the study of the receptor, and serve as a means for the isolation and cloning of the encoding genes.