Early detection in saliva of polypeptides associated to isoproterenol-induced mouse parotid hypertrophy

Chronic administration of isoproterenol (IPR) results in a marked hypertrophy and in the induction of a group of putative proline-rich polypeptides in the mouse parotid glands. Some of these polypeptides (pps C-G) have been considered as molecular markers of the parotid gland enlargement. Given the secretory character of polypeptides C-G, the polypeptide composition of mouse saliva was used to monitor the IPR-induced salivary gland hypertrophy. Whole saliva was collected after an oral administration of pilocarpine (PIL). Under those conditions, PIL provoked a massive salivary secretion both in normal control mice and during the whole course of the IPR-induced gland enlargement. Striking changes in the polypeptide composition of saliva obtained from chronically IPR-stimulated animals were observed. Those changes consisted basically in the appearance and progressive increase in concentration of parotid polypeptides C-G and in the progressive diminution in concentration of a couple of normal salivary polypeptides (polypeptides A-B). The appearance of new polypeptides in saliva could be established unequivocally within the 24 h following the trophic adrenergic stimulation. On the other hand, salivary polypeptides induced in response to a single administration of IPR could be demonstrated as late as 7-9 days after the stimulation. Accordingly, detection of parotid polypeptides C-G in PIL-produced saliva obtained from IPR-stimulated mice has proved to be a highly advantageous method to evaluate salivary gland hypertrophy both at very early stages after the trophic stimulation and late after the occurrence of the trophic episode.     

Induction of DNA synthesis by dichloroisoproterenol without initial rise of the cAMP level in the parotid gland of mouse

The increase in the cAMP concentration in the parotid gland of mouse, observed shortly after administration of isoproterenol, was not detected after dichloroisoproterenol, a β-adrenergic blocking agent. The latter compound, however, could induce DNA synthesis which was comparable to that induced by IPR in several respects. The results strongly suggest that the initial rise of cAMP concentration in the parotid gland after IPR injection is not related directly to the initiation of the stimulated DNA synthesis.     

Control of RNA synthesis by chromatin proteins.

The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA.     

Cyclic AMP mediated increased amino acid transport: effect of isoproterenol administration.

A single injection of isoproterenol (IPR) stimulates cell proliferation in rodent salivary glands after a lag period of about 24 hrs. Among the many events occurring prior to stimulated DNA synthesis, there is an early increase in cAMP levels and elevated transport of amino acids into the parotid gland. Amino acid transport is also elevated in liver and pancreas, tissues not induced to proliferate by IPR. IPR-stimulated alpha-aminoisobutyric acid (AIB) transport in parotid, pancreas and liver is augmented by prior injection of theophylline and is mimicked by dibutyryl cAMP. In all three tissues, changes in cAMP levels and subsequent increases in AIB transport appear to be closely related events. Since only the parotid gland is stimulated to grow after IPR injection, amino acid transport and growth would not appear to be directly related.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Cell proliferation in the mouse parotid gland after unilateral parotidectomy.

Unilateral parotidectomy with or without total submandibulectomy has been used to induce cell proliferation in mouse parotid gland. Maximum DNA synthesis and mitosis were recorded four and five days after the operation. The double operation increased the proliferative response. Such proliferative stimulii was not accompanied by secretion and was sex independent. On the other hand, the response decreased with age. RNA and protein synthesis inhibitors showed that the stimulation of DNA synthesis depends on early protein synthesis, which seems to be synthesized on a preexisting template.     

The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro

The larval salivary gland of Drosophila melanogaster synthesises a complex secretion, known as ‘glue’. which is secreted at puparium formation and then cements the puparium to its substrate. This secretion is made during the third larval instar and is stored in the gland cells as large granules. A few hours before puparium formation it is secreted into the gland's lumen by exocytosis. This process is induced by ecdysone and can be studied in vitro. Secretion is initiated about 3.5 hr after exposure of glands to ecdysone and is complete by 8 hr. The effects of varying the ecdysone concentration, of inhibitors of RNA or protein synthesis, and of withdrawing the hormone at various times after initial exposure on the process of secretion have been studied. We conclude that some event(s) occurring during the first 3 hr exposure to ecdysone is necessary to initiate secretion of the glue into the gland lumen. The possible relationship between this event(s) and the ecdysone induced changes in gene activity (puffs) which occur in the salivary glands at the same time is discussed.     

Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

Effects of epidermal growth factor on the syntheses of DNA and polyamine in isoproterenol-stimulated murine parotid gland

The effects of epidermal growth factor (EGF) on isoproterenol (IPR)-stimulated DNA synthesis and the activities of the rate limiting enzymes of polyamine synthesis (ornithine and S-adenosylmethionine decarboxylases) in parotid glands were investigated in vitro in cultured rat parotid explants and in vivo in submandibulectomized mice (mice after bilateral removal of the submandibular and sublingual glands). When the explants were cultured on siliconized lens paper floating on chemically defined synthetic medium, IPR caused the increases of both tissue cAMP level and the two decarboxylase activities in the prereplicative period and the stimulation of DNA synthesis with similar time courses to those observed in vivo. Dibutyryl cyclic AMP (DBcAMP) also increased the enzyme activities, but not DNA synthesis. EGF (1-2 ng/ml) had little effect on the IPR- and DBcAMP-dependent increases of amylase secretion and the enzyme activities, but it markedly enhanced IPR-stimulated DNA synthesis. Moreover, increase in DNA synthesis by DBcAMP was clearly observed in the presence of EGF when the explants were treated with this nucleotide analogue only during the early prereplicative period. In in vivo experiments, IPR-dependent increase in DNA synthesis was less in submandibulectomized mice than in intact animals. This decreased response to IPR of DNA synthesis was completely reversed by administration of EGF, though EGF alone did not induce either the enzymes or DNA synthesis. In submandibulectomized mice, although increases in the enzyme activities 8 h after injection of IPR were lower and they were significantly reversed by EGF, the activities at 12 h and the changes in polyamine levels at 8 and 12 h were almost the same as those in intact mice and were not affected by EGF treatment. These results obtained in vitro and in vivo suggest that EGF participates in the maximal response of IPR-dependent DNA synthesis but is not involved in the change of polyamine synthesis induced by IPR in murine parotid glands.     

Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.     

SECRETORY PROTEIN SYNTHESIS IN THE STIMULATED RAT PAROTID GLAND : Temporal Dissociation of the Maximal Response from Secretion

Administration of the β-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [¹⁴C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [¹⁴C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.     

Inhibition of cyclocytidine-induced enlargement of parotid and heart by propranolol or sympathectomy.

Enlargement of salivary glands and heart of mouse as well as rat is caused by chronic administration of the antitumor agent cyclocytidine. In rat, the effects are maximal within 3 days, and are reversible, but with heart, not readily so. The organ enlargement, in both species, is the result of an action of the cyclocytidine involving beta adrenergic receptors since administration of propranolol 20 min prior to injection of cyclocytidine prevented the enlargement as well as the increases in nucleic acids associated with the enlargement. These beta adrenergic effects appear mediated through indirect actions of cyclocytidine that probably involve release of norepinephrine from postganglionic nerve fibers. This conclusion is based on the findings that parotid sympathectomized for 5 days does not exhibit the same secretory responses and glandular enlargement with cyclocytidine administration that are observed in the innervated mate. For example, amylase activity of the innervated gland was reduced from 525 mg/mg of gland (unstimulated parotid) to approximately 190 two hr after administration of 500 mg/kg of cyclocytidine, whereas that of the sympathectomized gland was unchanged (510 mg/mg). In addition, cyclocytidine caused only a small increase in weight and nucleic acid content of the sympathectomized parotid, whereas the innervated mate exhibited the usual marked increases in all parameters (weight and RNA were twice as great as those of untreated rats). Guanethidine and bretylium were used in conjunction with cyclocytidine to provide additional evidence for the indirect beta adrenergic action of cyclocytidine on heart and parotid gland.     

Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat

Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands.The specific binding of the labelled beta-adrenergic antagonist [³H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [³H]-dihydroalprenolol compared with normal tissue.Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [³H]dihydroalprenolol binding sites during isoproterenol-induced growth.Previously-described differences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands.

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands. The specific binding of the labelled beta-adrenergic antagonist [3H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [3H]-dihydroalprenolol compared with normal tissue. Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [3H]dihydroalprenolol binding sites during isoproterenol-induced growth. Previously-described di-ferences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.