Alternative splicing model for the synthesis and secretion of the 20 kilodalton form of rat growth hormone

The characterization of a 20 kilodalton (20 kD) variant of rat growth hormone is reported. The 20 kD variant from rat pituitary gland extracts was identified on Western immunoblots of polyacrylamide gels. It was also shown that pituitary tissue maintained in culture secretes the 20 kD form. A rat growth hormone cDNA fragment was used as a probe in S1 nuclease mapping experiments of rat pituitary poly (A) mRNA to detect the presence of two growth hormone mRNAs in the rat pituitary gland. The protected mRNAs correspond to the predicted sizes that would encode the 22 kD and 20 kD forms of growth hormone. The site of variation between the mRNAs maps to a potential alternative 3' splice site in the 5' end of exon 3 of the coding sequence. The results support the hypothesis that the 20 kD variant in rat is the product of an mRNA alternatively spliced in exon 3, as is the case for the human growth hormone.     

Adenohypophyseal and hypothalamic GABA B receptor subunits are downregulated by estradiol in adult female rats

Gamma-aminobutyric acid (GABA) participates in neuroendocrine regulation. Since steroid hormones have been shown to modulate the GABAergic system, here we evaluated the effect of chronic in vivo estradiol administration on GABA B receptor (GABA(B)R) expression. GABA(B1) and GABA(B2) subunits were analyzed by Western Blot and RT-PCR, in hypothalami and anterior pituitaries of adult female rats: a) treated for 1 week with estradiol-valerate (a single dose of 100 mug /kg: E1), b) implanted with a 10 mg pellet of estradiol-benzoate for 5 weeks (E5) or c) on proestrous (P), d) ovariectomized (OVX). Pituitary GABA(B)R levels were correlated to a biological effect: baclofen, a GABA(B)R agonist, action on intracellular calcium titers ([Ca(2+)](i)) in pituitary cells. E5 pituitaries showed a significant decrease in the expression of GABA(B1) and GABA(B2) mRNAs compared to P. The GABA(B1a) splice variant of GABA(B1) was always more abundant than GABA(B1b) in this tissue. Similar to the pituitary, hypothalamic GABA(B1) and GABA(B2) mRNAs decreased in E5; this was confirmed at the protein level. In the hypothalamus GABA(B1b) was the main variant expressed in P rats, and was the one significantly sensitive to estradiol-induced decrease, as determined by Western Blots. Castration did not modify GABA(B)R expression with regards to P in either tissue. In P pituitary cells baclofen induced a decrease in [Ca(2+)](i), in contrast this effect was lost in E5 cells. We conclude that chronic estradiol treatment negatively regulates the expression of the GABA(B)R subunits in the pituitary and the hypothalamus. This effect is coupled to a loss of baclofen action on intracellular calcium in pituitary cells.     

The molecular basis for a cytosolic malic enzyme null mutation. Malic enzyme mRNA from MOD-1 null mice contains an internal in-frame duplication that extends the coding sequence by 522 nucleotides

Many tissues from wild type mice express cytosolic malic enzyme activity and contain two mRNAs (2.0 and 3.1 kilobases (kb)) that encode a single 64-kDa malic enzyme subunit polypeptide. MOD-1 null mutant mice lack cytosolic malic enzyme activity but express 2.5- and 3.6-kb mRNAs that hybridize with wild type malic enzyme cDNAs and are induced in liver by a starvation/carbohydrate refeeding regimen. To investigate the basis of the MOD-1 null mutation, a lambda gt11 cDNA library was constructed using mRNA from the livers of induced MOD-1 null mice as a template. A recombinant phage with a 2-kb insert was isolated by screening with wild type malic enzyme cDNA probes. The subcloned insert exhibited an atypical (non-wild type) restriction pattern and was subjected to sequence analysis. MOD-1 null malic enzyme cDNA contains an internal tandemly duplicated sequence that corresponds to nucleotides 1027-1548 in the coding region of wild type murine malic enzyme cDNA (Bagchi, S., Wise, L. S., Brown, M. L., Bregman, D., Sul, H. S., and Rubin, C. S. (1987) J. Biol. Chem. 262, 1558-1565). An open reading frame is retained throughout the duplicated sequence. The discovery of a 522-nucleotide in-frame duplication accounts for the increased size of MOD-1 null malic enzyme mRNAs and suggests that a variant malic enzyme polypeptide that is 19 kDa larger than the wild type subunit might be found in mutant mice. Western immunoblot analysis disclosed that MOD-1 null liver cytosol contains an 82-kDa protein that is recognized by anti-malic enzyme antibodies. Under stringent conditions, an anti-sense 32P-oligonucleotide that spans the abnormal junction between the reiterated sequences hybridized with the 2.5 and 3.6-kb MOD-1 null malic enzyme mRNAs but failed to form stable complexes with wild type malic enzyme mRNAs. Thus, both MOD-1 null malic enzyme mRNAs contain the duplication deduced from cDNA sequence analyses. The MOD-1 null mutation might originate from an unequal crossover between homologous regions of two different introns in the malic enzyme gene, thereby causing the duplication of one or more exons.     

Identification of a novel pituitary-specific chicken gonadotropin-releasing hormone receptor and its splice variants

In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%-65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.     

Antisense Strategy Unravels a Dopamine Receptor Distinct from the D2 Subtype, Uncoupled with Adenylyl Cyclase, Inhibiting Prolactin Release from Rat Pituitary Cells

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [³H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.     

Effect of estradiol 17-beta on LH subunits and prolactin mRNAs expression in the pituitary of old female rats

OBJECTIVES: The aim of the study was to examine susceptibility of the pituitary gland to estrogenic impulse in old, noncycling rats by measurement of steady state level of mRNAs encoding LH subunits a and b and mRNA for PRL. METHODS: 22-month-old rats were ovariectomized and after one week they were subcutaneously implanted with silastic tubing filled with oil or with estradiol 17-beta. Pituitary alpha, LHbeta and PRL mRNAs content and serum LH and PRL concentration was determined. RESULTS: The effect of E (2)treatment was manifested by the significant increase in the weight of the uterus and pituitary gland as well as by elevation of total pituitary RNA (109%, 60% and 78%, respectively; p<0.001). No significant changes (p>0.05) in serum LH concentration were observed, while levels of mRNAs encoding alpha and LH-beta subunits were lowered by 54% (p<0.05) and 96% (p<0.01), respectively, in the rats subjected to E(2) stimuli. No direct correlation between synthesis and release of LH in E(2) treated old rats was observed. The blood PRL concentration and the pituitary level of PRL mRNA increased up to 2,000% and 1,300%, respectively (p<0.001). Spontaneous pituitary adenoma was observed in about 30% of the rats, irrespective of treatment. CONCLUSIONS: These data show that in old rats estrogenic stimulus can effectively diminish both pituitary LH subunits mRNAs as well as stimulate pituitary PRL mRNA level indicating that the E(2)-dependent processes involved in the regulation of corresponding genes are still functional.     

Sexual dimorphism in amino acid compositions of mouse prolactin

Sexual dimorphism was found in amino acid compositions and immunogenecity of variant types of prolactin (PRL) purified from the pituitary gland of normal adult C57BL mice by a high performance liquid chromatography. From the pituitary gland of female mice, three female variant types of PRL were isolated, whereas from the pituitary gland of male mice, two male variant types of PRL (M1-PRL and M3-PRL) and a female variant type of PRL (M2-PRL) were obtained. The amino acid composition of M3-PRL was different from any of female variant types which were very similar to each other and contained less varine, isoleucine, leucine, tyrosine and lysine but more alanine than the latter. Immunoreactivity of any of female variant types of PRL against anti-PRL serum was 100%, whereas that of M1-PRL was as much as 85% and that of M3-PRL was nearly undetectable.     

Expression of several amplified genes in an adenylate-deaminase overproducing variant of Chinese hamster fibroblasts

Unstable variants with increasing amounts of adenylate-deaminase (AMPD) have been stepwise recovered from Chinese hamster fibroblasts plated in selective medium containing increasing coformycin concentrations; several polypeptides accumulate in the variants in parallel to AMPD: they are no longer detectable in cells which reverted to the wild-type enzyme level. We report here the molecular cloning of cDNA sequences complementary to mRNAs coding for four such polypeptides. The plasmidic probes have been exploited to characterize their complementary mRNAs and to quantify the copies of these cognate genes in a variant and in two revertant clones. The results show that different mRNAs code for the four polypeptides; their accumulation is accounted for by amplification of their specific genes; these observations suggest that cells overproducing AMPD are characterized by the presence of amplification units comprising several expressed genes.     

Regulated Capture by Exosomes of mRNAs for Cytoplasmic tRNA Synthetases

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.     

Effects of acid water exposure on plasma cortisol, ion balance, and immune functions in the "cobalt" variant of rainbow trout

This study was undertaken to examine physiological responses to acidification of environmental water in the "cobalt" variant of rainbow trout (Oncorhynchus mykiss), which exhibits malformation of the pituitary, by following changes in plasma levels of cortisol and electrolytes, blood pH, gill Na(+), K(+)-ATPase activity, and immune functions after exposure to acid water (pH 4.5). Resting levels of plasma cortisol and lysozyme were significantly lower in the cobalt variant than in the normal trout, whereas plasma ceruloplasmin was significantly higher in the cobalt variant, suggesting that some endocrine factors, lacking or deficient in the cobalt variant, are important for the regulation of its immune functions. Blood pH was slightly but significantly lower in the cobalt variant at rest. After exposure to acid water for 24 h, both the normal trout and cobalt variant showed a significant elevation in plasma cortisol, although the increased level in the cobalt variant was still lower than that in the normal trout transferred to neutral water. No differences were seen in blood pH, plasma electrolytes, and gill Na(+), K(+)-ATPase activity between the normal trout and the cobalt variant, indicating that the cobalt variant regulates ion balance when exposed to acid water, despite malformation of the pituitary. Although the normal trout showed a reduction in plasma lysozyme level after acid exposure, there was no significant change in the cobalt trout. Adverse effects of pituitary malformation on ion balance and immune functions may be compensated by extrapituitary factors in the cobalt variant when it is exposed to acid water.     

Complementary expression of IGF-II and IGFBP-5 during anterior pituitary development

The specification of the five individual hormone-secreting cell types in the anterior pituitary requires a series of sequential cell fate decisions. We have immortalized cells at several stages along this pathway of pituitary differentiation. Here, we present analysis of differences in gene expression between an anterior pituitary precursor cell line, alphaT1-1, and an immature gonadotrope cell line, alphaT3-1, identified by using cDNA subtraction. Messenger RNA expression of members of the insulin-like growth factor signaling system, IGF-II and IGFBP-5, was found in the alphaT1-1 precursor cell line, but not in the more differentiated cell line, alphaT3-1. This inferred stage specificity was confirmed in the mouse embryo by using in situ hybridization on embryonic days e10.5 through e18.5. Expression of IGF-II and IGFBP-5 mRNAs was both temporally and spatially regulated during pituitary development. IGF-II was highly expressed in the epithelium surrounding Rathke's pouch at e10.5, while IGFBP-5 expression was restricted to the adjacent oral epithelium. At e11.5 (represented by alphaT1-1), IGF-II was expressed throughout the pouch, but was coexpressed with IGFBP-5 and alpha-subunit in the ventral portion of the pouch epithelium. On e12.5, the two mRNAs were expressed in opposing dorsoventral (IGF-II) and ventrodorsal (IGFBP-5) patterns, with IGF-II excluded from the rostral, alpha-subunit-expressing region. A decrease of both mRNAs was observed at e14.5 (equivalent to alphaT3-1), with IGF-II levels low and IGFBP-5 concentrated in the anterior pituitary rostral tip. These findings suggest that the timing of IGF-II expression and regulation of its accessibility by IGFBP-5 may play a role in anterior pituitary differentiation, survival, and/or proliferation.