Effects of ionizing radiation and beta-adrenergic stimulation on the expression of early response genes in rat parotid glands.

There is little known about the regulation of gene expression in rat parotid glands after exposure to ionizing radiation. The present studies investigate the effects of in vivo ionizing radiation, with subsequent stimulation of beta-adrenergic receptors by isoproterenol, on parotid gland function and on the expression of the early response genes, c-fos, c-jun, and jun B. Ionizing radiation diminished parotid gland weight and saliva output. Treatment of irradiated rats with isoproterenol increased the gland weight to levels similar to those in nonirradiated rats. However, such treatment had no effect on saliva output as indicated by measurements of parotid salivary flow rate. Irradiation alone increased the expression of c-fos, c-jun, and jun B. The combination of irradiation and isoproterenol had an additional effect on the levels of c-fos and jun B mRNAs and proteins particularly at earlier experimental times (1 to 8 h). Isoproterenol alone induced high levels of c-fos and jun B mRNA but not of c-jun mRNA. However, c-jun mRNA was induced markedly by radiation and 8 h of isoproterenol treatment, indicating a combined effect on c-jun gene expression. These observations suggest that the expression of the proto-oncogenes c-fos, c-jun, and jun B is probably regulated through differential signal transduction pathways which may be activated by these external stimuli and may be associated with functional changes induced in the rat parotid gland by ionizing radiation and by ionizing radiation and isoproterenol.     

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of beta-adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.     

A single mouse alpha-amylase gene specifies two different tissue-specific mRNAs

The alpha-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5' non-translated sequences: the 5' terminal 158 nucleotides of the major liver alpha-amylase mRNA are unrelated to the 5' terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5'terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver alpha-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5' terminal 50 nucleotides of the salivary gland alpha-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver alpha-amylase mRNAs are transcribed from the same gene (Amy1A). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amy1A. Rather, these data indicate that the salivary gland and liver alpha-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.     

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.     

Tissue-specific expression of mouse α-amylase genes: Nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland

We have determined the nucleotide sequence of two different mouse α-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively, are the major α-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5′ noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict α-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland α-amylase mRNA is not initiated at the AUG codon nearest the 5′ terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.     

Characterization of primary translation products from ovine and rat salivary gland mRNAs

Ovine and rat salivary gland mRNAs have been prepared and their translation products characterized. A 60 kD translation product from ovine submaxillary and sublingual gland mRNAs is identical in mass to the ovine apomucin. Two additional ovine translation products, 25 and 40 kD, are specific to mucin-producing salivary glands. Four rat mRNA translation products are encoded by mucin-producing salivary glands (38, 44, 67, 69 kD). These polypeptides were not detected in the parotid gland mRNAs, a serous gland. Each of these products has a high level of [3H]serine incorporation, a characteristic of mucins. The nature of these products suggests that they are mucins or mucin-like and that their molecular weights should approximate that of the corresponding apomucins.     

Alpha-lactalbumin acts as a bimodal regulator of rat parotid acinar cell growth

Isoproterenol, a beta-adrenergic receptor agonist, causes hypertrophy and hyperplasia of the rat parotid gland. The stimulation of parotid acinar cells to a growth phase is accompanied by a cell surface localization of the enzyme 4 beta-galactosyltransferase. Alpha-lactalbumin, a specific modifier protein for 4 beta-galactosyltransferase, when given subsequent to the initiation of isoproterenol treatment and the commencement of parotid enlargement, resulted in a termination of gland hypertrophy and DNA synthesis. Gland size did not, however, return to control levels with the continued injection of isoproterenol and alpha- lactalbumin. In contrast, the injection of alpha-lactalbumin in neonatal rats (7-14 days post-partum) stimulated parotid gland hypertrophy and DNA synthesis. This treatment also lead to the precocious expression of the major parotid gland salivary enzyme, amylase.     

Differential expression of isoproterenol-induced salivary polypeptides in two mouse strains that are congenic for the H-2 histocompatibility gene complex

Two inbred mouse strains, A/Snell and A.Swiss, which were produced as congenic with regard to the H-2 histocompatibility gene complex, are homozygous for two different groups of isoproterenol-induced salivary polypeptides (IISP). These polypeptides, which have been considered as markers of the hypertrophic growth of the parotid acinar cells, are members of the complex family of salivary proline-rich proteins (PRP) on the basis of both their massive accumulation in the parotid acinar cells in response to chronic isoproterenol, secretory character, high solubility in trichloroacetic acid and metachromatic staining by Coomassie blue. IISP expressed in both mouse strains were identified by unidimensional SDS-polyacrylamide electrophoresis and Coomassie blue staining both in parotid gland homogenates and in whole salivas obtained from mice repeatedly stimulated at 24-h intervals with isoproterenol. Parotid glands from 40 mice (20 A/Snell and 20 A.Swiss) and salivas from 270 mice (200 A/Snell and 70 A.Swiss) were analyzed. One of the congenic strains (A/Snell) expressed five IISP (Mr 65, 61, 51.5, 38, and 37 kDa) and the other strain (A.Swiss) expressed six IISP (Mr 59, 57, 54.5, 46, 36, and 34 kDa). No inter-individual intra-strain variations were observed, thus defining strain-associated patterns of IISP (PRP).     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.     

Unexpected beta adrenergic effects of the antitumor agent, cyclocytidine, on rat salivary glands.

In addition to its potent antileukemic properties, cyclocytidine has a sialogogue action that depends on stimulation of beta adrenergic ereceptors of salivary glands. Furthermore, when chronically administered (for 3 days), cyclocytidine caused enlargement of parotid and submaxillary glands and heart that resembled the hypertrophy caused by chronic isoproterenol administration. The salivas evoked by cyclocytidine also closely resembled those evoked by isoproterenol, and were extremely viscous, and high in K+, (121 plus or minus 5.6, for submaxillary, and 42 plus or minus 2.9, for parotid), low in flow rate (0.007 mg/min times mg) and parotid saliva contained high concentrations of amylase (805 plus or minus 33 mg/mg gland). Cyclocytidine also caused marked emptying of parotid gland amylase. The cyclocytidine-induced salivary flow and gland emptying of amylase were prevented for 90 min when propranolol (but not dibenzyline or atropine) was administered prior to injection of the cyclocytidine. In addition, when the superior cervical ganglion was acutely removed, administration of cyclocytidine elicited salivary flow from the denervated as well as the innervated glands. These findings suggest that cyclocytidine does not affect salivary glands through indirect central or ganglionic actions. Cyclocytidine action does not exclusively involve beta receptors, since even in the presence of propranolol, secretory flow was evident after 90 min but when dibenzyline was given with the propranolol, complete blockade of cyclocytidine-stimulated saliva was effected. The dominant effect is, however, a beta adrenergic one. The undesirable side effects of cyclocytidine (parotid pain, postural hypotension, and cardiac hypertrophy) probably stem chiefly from its beta adrenergic properties and might be eliminated (or at least modified) by administration of propranolol with the cyclocytidine.     

Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.     

Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

In vivo changes in free choline level induced by autonomic agonists in mouse organs, including three major salivary glands

Whether free choline levels are changeable in vivo in response to different types of autonomic agonists was examined in several mouse organs. Upon one subcutaneous injection of isoproterenol, phenylephrine and pilocarpine, choline levels in whole organ decreased, increased and decreased, respectively, in various organs within 30 min and returned to initial levels in a day. In the three major salivary glands, a delayed choline elevation also appeared on day 2 after one isoproterenol injection and subsided by day 6. Only in the three salivary glands more choline was accumulated after 10 once-a-day injections of isoproterenol than after one isoproterenol injection. Neither phenylephrine nor pilocarpine induced comparable choline accumulation in any organs examined. Isoproterenol injection repeated at a 2-day interval augmented the subsequent, delayed choline elevation. Examination with dobutamine and the adenylyl cyclase activator 6-(3-dimethylaminopropionyl)forskolin suggested that isoproterenol-induced immediate choline lowering was down-stream of cAMP synthesis and linked to cAMP more tightly than the choline accumulation, though both choline changes occurred via beta1-adrenergic receptors. Choline levels in the salivary glands also changed depending on the form of diet given and particularly in the parotid gland in parallel with gland weights. These results provide the first evidence for the autonomic control of intracellular choline levels; intracellular choline levels might be an integral part of the autonomic signalling pathway.     

Differential expression of type-specific c-abl mRNAs in mouse tissues and cell lines.

The mammalian c-abl proto-oncogene produces mRNAs with 5' heterogeneity from two distinct promoters and the alternative splicing of variable 5' exons. By using quantitative RNase protection assays, the relative abundance of two major c-abl mRNAs, type I and type IV, in several mouse tissues and cell lines has been determined. Our results demonstrate that the level of type IV c-abl mRNA is rather constant, whereas that of the type I mRNA varies over a 10-fold range in different tissues and cell types. This finding has interesting implications for the function of the two c-abl proteins.     

Strain-specific differences in the proline-rich proteins and glycoproteins induced in rat salivary glands by chronic isoprenaline treatment.

Parotid and submandibular glands were isolated from five strains of rat after chronic injection of the beta-adrenergic receptor agonist isoprenaline (isoproterenol). The glands were observed to have undergone a marked increase in wet weight, owing to hypertrophy and hyperplasia. The 100 000 g soluble fraction of gland cell lysates were extracted with 10% (w/v) trichloroacetic acid, and the soluble material subsequently analysed by SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. By this procedure, evidence was obtained for the induction, in isoprenaline-treated parotid and submandibular glands, of proline-rich proteins with apparent Mr values ranging from 20 000 to 40 000. Heterogeneity was evident in the proteins produced for a specific gland between the rat strains, although the amino acid compositions were the same. Products from induced mRNAs translated in vitro had similar mobilities in SDS/polyacrylamide gels, despite the apparent difference in mobility of trichloracetic acid-extracted proline-rich proteins from the various strains. Strain-specific differences were noted for the proline-rich glycoproteins from control salivary glands as well as those induced as a consequence of isoprenaline treatment. Although the glycoproteins had similar amino acid compositions, there was considerable heterogeneity in the carbohydrate compositions for these proteins, suggesting that the differences were the result of post-translational modifications during glycosylation. Induction of the increased activity of the Golgi membrane marker enzyme UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) occurred to the same extent in the parotid glands of all strains examined. There was no change in the specific activity of a second enzyme, UDP-galactose:N-acetylgalactosaminyl-protein 3 beta-galactosyltransferase (no EC designation).     

Coordination of murine parotid secretory protein and salivary amylase expression.

PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.