Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in the submaxillary gland

Synthesis of alpha 2u globulin, previously thought to occur only in the male rat liver, has now been demonstrated in the submaxillary salivary gland. Unlike liver, submaxillary synthesis of alpha 2u globulin mRNA is constitutive--that is, independent of the endocrine state, age and sex. Liver and submaxillary alpha 2u globulin mRNAs are of similar size, and their 5' ends map to the same region of the gene. Isoelectric focusing of in vitro translation products revealed that submaxillary mRNA encodes a more acidic subset of alpha 2u globulins than does liver. Salivary alpha 2u globulin mRNA manifests 5% nucleotide divergence, encoding 20 amino acid substitutions, which specifies a more acidic polypeptide than its hepatic counterpart. Thus the liver and submaxillary gland synthesize alpha 2u globulin from different sets of genes that are subject to very different developmental and hormonal control.     

Tissue-specific expression of the rat alpha 2u globulin gene family.

The rat alpha 2u globulin gene family encodes approximately 20 low-molecular-weight (20,000) proteins with pIs ranging from 4.5 to 7.9. alpha 2u globulin protein isoforms were detected in the liver and in the submaxillary, lachrymal, preputial, and mammary glands of Sprague-Dawley rats. The hormonal and developmental regulation of alpha 2u globulin synthesis in each of these tissues was unique, and it appears that different alpha 2u gene sets were transcribed in the various tissues.     

Identification of alpha-2u globulin in the rat preputial gland by MALDI-TOF analysis

The low molecular mass proteins found in the pheromonal sources such as urine, saliva, glandular secretion etc have been reported as ligand carriers for the processes of chemocommunication in mammals. The preputial gland plays an important role in the production of olfactory signals for pheromonal communication. Thus, in the present study, alpha-2u globulin having molecular mass of 18 kDa has been identified in the preputial gland of Norway rat (Rattus norvegicus) by in-gel trypsin digestion and analyzing the resulting peptides by MALDI-TOF. Since preputial gland is one of the major pheromonal sources in rat, the results suggest that alpha-2u globulin might act as a carrier for hydrophobic odorants of preputial gland.     

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.     

Evaluation of a reservoir in the main excretory duct of rat submaxillary gland.

Cannulation of salivary gland main excretory duct at its oral opening is routinely used for collecting fluid, in situ, from the luminally perfused duct, or saliva from the stimulated gland. For perfusion of the main excretory duct, in situ, or for saliva collection, rat submaxillary gland is often the organ of choice, since electrolyte transport occurs at high rates both in the whole gland and in the main excretory duct. Recently, it has been reported that there is a pouchlike dilatation of the main excretory duct at its oral end, and that this dilatation may serve as a fluid reservoir. Because of possible effects of such a reservoir on measurements of electrolyte transport by the whole gland or the main duct segment, the size and form of the reservoir have now been examined. For this, techniques of histology, radiography, and microcatherization were employed. It was found that, while the functional volume of the reservoir exceeds that of the main duct proper, the time needed for displacement of reservoir fluid by perfusate or saliva would probably be only on the order of 1-3 min at higher rates of saliva or perfusate flow. Therefore, if adequate allowance is made for equilibration time, collection of saliva or luminal perfusate by oral cannula seems justified.     

Age-dependent reversal of the lobular distribution of androgen-inducible alpha 2u globulin and androgen-repressible SMP-2 in rat liver.

Hepatocytes situated at pericentral and periportal zones of the liver lobule show differences in the expression of several liver-specific genes, such as androgen-inducible alpha 2u globulin and androgen-repressible senescence marker protein-2 (SMP-2). A marked temporal difference in the expression of these two androgen-regulated genes has also been observed. The liver of the pre-pubertal male rat is insensitive to androgen, and during this period hepatocytes synthesize only SMP-2. During young adult life (greater than 40 days), the liver becomes androgen sensitive and concomitant synthesis of alpha 2u globulin and repression of SMP-2 occur. In the senescent male rat (greater than 750 days), the liver again becomes androgen insensitive when the decline in alpha 2u globulin is accompanied by an increase in SMP-2 synthesis. In this article we present results to show a correlation between the temporal and spatial (intralobular) changes in the expression of the androgen-inducible alpha 2u globulin and the androgen-repressible SMP-2 in rat hepatocytes. Results indicate that the temporal changes in hepatic androgen sensitivity are dictated by the intralobular location of the hepatocytes. Hepatocytes located around the central vein (pericentral/perivenous) may benefit from a paracrine advantage for the expression of a subset of genes, including the gene for the androgen receptor.     

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.     

Induction of alpha 2u globulin mRNA by phenobarbital in rat liver: characterization of a cDNA clone

A clone has been selected from a cDNA library previously constructed from phenobarbital pre-treated rat liver polysomal poly(A)+ RNA, which was reverse transcribed. The double-stranded cDNA was inserted by GC homopolymeric tailing in the Pst I site of pAT 153, and further cloned in E. coli HB101. This clone, called 2A9, corresponds to a mRNA whose concentration is increased five fold 16 h after phenobarbital treatment. Its length is 1200 nucleotides as revealed by RNA dot and Northern blot analysis respectively. The two strands of a 450 bp fragment from the 2A9 580 bp double-stranded cDNA insert have been sequenced and proven to correspond to alpha 2u globulin mRNA. It shows one single bp difference from the sequence previously published by Unterman et al. (1981, PNAS, 78, 3478). Thus, alpha 2u globulin, a hormone regulated gene product, is inducible by phenobarbital.     

Pyroglutamate aminopeptidase in rat submaxillary gland.

A significant amount of pyroglutamate aminopeptidase (PGAP) activity was found to be present in 27,000 x g supernatant of rat submaxillary gland, maximum activity being at pH 6.5. EDTA stimulated the enzyme activity by 95% at pH 8.0 while at pH 6.5 it did not have any significant effect. On comparison of its properties submaxillary PGAP appears to be different from brain, pituitary and other reported PGAPs. Submaxillary PGAP could also catalyze efficiently the formation of cyclo (His-Pro) from TRH. Cyclo (His-Pro) formation by submaxillary enzyme was more pronounced than that by liver PGAP.     

Ultrastructural immunocytochemical localization of calcitonin in the C cells of the rat thyroid

Using unlabeled antibodies and peroxidase-anti-peroxidase complexes, calcitonin was localized at the ultrastructural level in rat thyroid C cells. Calcitonin was present mainly in secretory granules of the cells. A less intense positive reaction was noted in the cytoplasm surrounding the secretory granules.     

Ultrastructural immunocytochemical localization of calcitonin in the C cells of the rat thyroid

Summary Using unlabeled antibodies and peroxidase-anti-peroxidase complexes, calcitonin was localized at the ultrastructural level in rat thyroid C cells. Calcitonin was present mainly in secretory granules of the cells. A less intense positive reaction was noted in the cytoplasm surrounding the secretory granules.     

Hormonal regulation of the hepatic messenger RNA levels for alpha2u globulin.

The messenger RNA rat alpha2u globulin has been identified and quantitated in a cell-free translational system derived from Krebs II ascites cells. Hepatic tissue of the mature male rats which normally produce alpha2u globulin was also found to contain a high level of alpha2u mRNA. Approximately 1.6 per cent of all poly(A) containing RNA of the adult male rat liver could be accounted for alpha2u messenger activity. Female rats do not produce alpha2u globulin and no alpha2u mRNA activity could be detected in the poly(A) containing RNA fraction obtained from the livers of these animals. However, androgen treatment to spayed female rats was found to induce the parallel appearance to both alpha2u globulin and its corresponding mRNA. Both hypophysectomy and adrenalectomy which are known to reduce the level of alpha2u globulin in the urine of male rats were found also to reduce the hepatic level of alpha2u mRNA. The results indicate that hormonal control of alpha2u globulin synthesis in rat liver is achieved primarily through regulation of its translatable mRNA level and that more than one hormone may participate in this regulation.     

Ultrastructural changes in the rat submaxillary gland following isoprenaline

Summary The ultrastructural appearance of acinar cells of the submaxillary gland have been examined at various time intervals following an injection of isoprenaline; the changes observed in previously untreated rats were compared with those in animals which had received differing periods of chronic treatment. The depletion and re-accumulation of secretory material was found to follow a similar time course irrespective of earlier treatment but there were quantitative differences in magnitude of response. An injection of the drug resulted in almost total depletion of secretory granules in previously untreated animals while in those which had received chronic treatment the depletion was less complete. This difference is discussed in relation to the decline in the hyperplasic response with prolonged drug treatment. The secretory granules showed better preservation of membranes and frequently contained dense configurations after chronic treatment.I am indebted to S. Glanvill, B. Mason, and D. O'Reilly for their skilled assistance.