Mouse Parotid Gland Research Articles

The Mouse Parotid Gland Preparation: An in vivo Pharmacological Tool

The effects of several autonomic agents on total protein, amylase, RNase, calcium, potassium, and sodium levels and also on flow rate of mouse parotid saliva were investigated during the process of evaluating the in vivo mouse parotid gland preparation as a pharmacological tool. Acetylcholine and pilocarpine were used as secretagogues and produced different sequential patterns of sodium and enzyme levels secreted in mouse parotid saliva. Additionally, propranolol treatment was able to alter amylase and sodium levels produced by pilocarpine stimulation significantly, as well as alter calcium levels produced by both acetylcholine and pilocarpine stimulation. Phentolamine treatment resulted in higher amylase levels with both acetylcholine and pilocarpine stimulation. The use of acetylcholine as a secretagogue allows one to perform in vivo anticholinergic experiments which alter flow rates without significantly altering protein or ion levels secreted in mouse parotid saliva.

Induction of proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and by tannins.

Glycoproteins which contain about 45 mol% proline were dramatically induced in mouse parotid and submandibular glands by isoproterenol treatment, but these unusual proteins were not detected in control animals. These acid-soluble substances were obtained by extracting tissues with 10% trichloroacetic acid, as reported previously for isolating proline-rich proteins from rat submandibular glands (Mehansho, H., and Carlson, D.M. (1983) J. Biol. Chem. 258, 6616-6620). Three major proline-rich glycoproteins were induced in parotid glands with apparent molecular weights of 66,000 (GP-66p), 45,000 (GP-45p), and 27,000 (GP-27p), whereas only one such protein was expressed by the submandibular glands (66,000 (GP-66sm]. Both GP-66p and GP-66sm contained about 19% carbohydrate with the following molar ratios, respectively; GalNAc, 1.0, 1.0; Gal, 1.6, 2.3; GlcNAc, 0.8, 1.1; sialic acid, 0.9, 1.9. The peptide chains of GP-66p and GP-66sm appear to be identical by amino acid compositions, glycopeptide analysis, and preliminary amino acid sequencing data. Northern blot analysis of RNAs from parotid glands of normal and isoproterenol-treated rats, probed with a 32P-labeled proline-rich protein cDNA, confirmed that control animals were devoid of mRNAs encoding these proteins and that isoproterenol treatment dramatically induced expression of these genes. Feeding sorghum high in tannins caused changes in the parotid glands similar to those observed upon isoproterenol treatment, as noted earlier with rats (Mehansho, H., Hagerman, A., Clements, S., Butler, L., Rogler, J., and Carlson, D.M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3948-3952). These glycoproteins have high affinities for tannins as demonstrated by competitive binding curves.

Analysis of sialograms: microradiograms of mouse parotid glands with reduced saliva excretion

Analysis of sialograms: microradiograms of mouse parotid glands with reduced saliva excretion

Activation of Ca2+-dependent Cl- and K+ conductances in rat and mouse parotid acinar cells.

Isolated acinar cells from rat and mouse parotid glands were studied with patch-clamp whole-cell current recordings. Acetylcholine (ACh) stimulation caused a transient inward current at a membrane potential of -70 mV, and a sustained outward current at a membrane potential of 0 mV, in quasi physiological Na+, K+ ion gradients, except the zero-Cl- ion gradient condition across the membrane. The reversal potential obtained from the ACh-evoked steady current was about -75 mV, in this ionic condition. When major Cl- ions of both the pipette and the bath solution were replaced, either by glutamate or by sulphate, only a large outward current was observed, at a membrane potential of -60 mV, in the presence of ACh. The addition of Ca2+-ionophore A23187 caused responses similar to those evoked by ACh. The reversal potential of A23187-induced current was close to the K+ equilibrium potential of -90 mV, in a Cl- -free condition. When K+-free NaCl solution was used in the pipette and the bath, A23187 caused only a large inward current, at a membrane potential of -60 mV. The reversal potential of A23187-evoked current was about -15 mV, in a symmetrical K+-free, NaCl condition. These results suggest that the ACh and A23187 activate Cl- as well as K+ conducting pathways via an increase in [Ca2+]i in the parotid acinar cells. The A23187-evoked large K+ current could not be explained solely by a rise in open probability of the channels.

Molecular cloning of mouse PSP mRNA

PSP is the most abundant translation product of mouse parotid glands where its production is co-ordinated with that of salivary amylase. The synthesis of these two proteins apparently is restricted to this tissue. In order to enable us to study common regulatory elements in the genes of the two proteins, double stranded cDNA, synthesized for parotid gland poly (A)+ RNA, was cloned. DNA sequencing of three clones complementary to the most abundant messenger indicated overlap and resulted in a total sequence of 867 nucleotides. Translation of this sequence revealed that at one end the amino acid sequence was the same as the N-terminal sequence of PSP. The sequence contains 60 nucleotides coding for part of or the complete signal peptide, 645 nucleotides coding for the PSP protein, and 162 nucleotides that apparently are not translated. Southern blot analysis suggests a simple structure for the PSP gene in mouse and man.

β-adrenergic stimulation of mouse parotid gland: Amylase activity and secretory granules during isoproterenol-induced mitosis

1. 1. This paper examines the mitotic activity of parotid glands of groups of mice sacrificed at 1 hr-intervals between 26 and 36 hr after being injected with dl-isoproterenol (IPR, 1 μmol/g, single dose, i.p.). 2. 2. Maximal mitotic activity occurs 35 hr after IPR injection; there is a concomitant rise of amylase activity (64% over control). 3. 3. Mitotic cells showed increased number of secretory granules ( N) with regard to interphasic cells. However, the increment of N is paralleled by an increment of the cell section area ( A); the N A ratio is thus maintained around 0.45 throughout. 4. 4. These findings suggest that during IPR-induced mitosis of mice parotid cells there is not a blockade of the biosynthetic pathways which lead to the appearance of secretory granules in the cell cytoplasm.

Carbonic anhydrase in mouse salivary glands and saliva: a histochemical, immunohistochemical, and enzyme activity study.

Mouse parotid gland and saliva were studied by histochemical, immunohistochemical, and activity measurements for carbonic anhydrase. Hansson 's histochemical reaction for carbonic anhydrase revealed positive enzyme activity in the parotid acinar cell cytoplasm and little or no reaction in the secretory granules. The luminal contents in all of the glandular duct systems also reacted positively, but the duct cells themselves were only weakly positive. Ultrastructural observations confirmed the light microscope histochemical localization and, in addition, revealed luminal content activity in intercellular ducts. Purified carbonic anhydrase isolated from mouse salivary glands was used to raise antibodies in rabbits. Localization of carbonic anhydrase by direct immunolabeling with fluorescein-coupled antibody and indirect immunoperoxidase labeling revealed enzyme localization on or in the acinar cell secretory granule membrane and not in the surrounding cytoplasm. The luminal contents of the intercalated and striated ducts were also strongly positive. Stimulation of salivary secretion with phenylephrine or pilocarpine increased the amount of carbonic anhydrase in saliva. Acetatazolamide and potassium cyanate inhibited carbonic anhydrase activity. Reasons underlying the discrepancy between the histochemical and immunolabeling localization of carbonic anhydrase are discussed. It is concluded that the parotid acinar cells of mice appear to be a significant source of carbonic anhydrase in saliva but its role is enigmatic.

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg 2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.

The effect of isoproterenol and cycloheximide on protein synthesis and growth in mouse parotid

Isoproterenol induces an increase in both cell size (hypertrophy) and cell number (hyperplasia) in the mouse parotid gland. The dose responses to isoproterenol of these two growth parameters are not identical because hypertrophy requires a severalfold lower dose. Cycloheximide injected 1 2 –1 hr after isoproterenol inhibits both growth responses but hyperplasia is more sensitive to this inhibitor. Isoproterenol depresses protein synthesis by about 50% during the first 6 hr after its administration, whereas cycloheximide (0.02–0.03 mg/g body wt) alone decreases protein synthesis by a similar amount but for much shorter periods. When cycloheximide is injected after isoproterenol the inhibition of protein synthesis is greater and the recovery does not follow the kinetics of cycloheximide-treated mice but parallels those of the isoproterenol-treated mice. Thus, isoproterenol appears to alter the organization and function of the acinar cells making the effects of cycloheximide more intense and prolonged than upon nonstimulated glands. Therefore, the effects of cycloheximide upon protein synthesis in the normal parotid cannot be extrapolated to glands that have been stimulated by isoproterenol. Mice treated with both cycloheximide and isoproterenol are completely refractory to the proliferative effects of a second isoproterenol injection for at least 4 hr and a full response is not restored until 14 hr later. This loss of response appears to be due to the initial isoproterenol administration rather than to the cycloheximide.

Sialic acid in mouse parotid plasma membranes: Its relationship with secretion and cell proliferation

A low dose of isoproterenol (1.5 nmoles/g body weight) induces secretion in mouse parotid glands while a high dose of this drug (670 nmoles/g body weight) provokes secretion and cell proliferation. Parotid plasma membranes were prepared from mice treated with the low or with the high dose of isoproterenol as well as from control mice. Peripheral and integral components were obtained from these membranes by using increasing concentrations of sodium deoxycholate. Sialic acid and protein content were measured into each fraction. A decrease in the sialic acid content in plasma membrane components extracted with low concentrations of sodium deoxycholate was found to be related to secretion. Removal of sialic acid from integral plasma membrane components which are extracted with higher concentrations of sodium deoxycholate seems to be related with induction of cell proliferation.

Effect of sodium ions on cyclic AMP and cyclic GMP levels in mouse parotid acini

The ability of the β-adrenergic agonist, isoproterenol, to elevate intracellular levels of cyclic-AMP (c-AMP) and cyclic GMP (c-GMP) in mouse parotid acini was dependent upon the extracellular sodium concentration. In the absence of extracellular sodium isoproterenol-stimulated c-GMP and c-AMP levels were significantly reduced; carbachol-stimulated c-GMP levels were not affected. Monensin, a sodium ionophore, mimicked the effects of isoproterenol in elevating c-GMP levels; this effect was abolished in the absence of extracellular sodium. Monensin did not mimic the effects of isoproterenol in elevating c-AMP levels. The data presented suggests that sodium ions may play a role in β-adrenergic regulation of cyclic nucleotide levels in mouse parotid gland and that the mechanisms involved in regulation of c-AMP and c-GMP levels appear to be different.

Phosphorylation of the same specific protein during amylase release evoked by beta-adrenergic or cholinergic agonists in rat and mouse parotid glands.

Stimulation of amylase secretion from the rat parotid gland by beta-adrenergic agonists is associated with a specific phosphorylation of three membrane-bound proteins designated as proteins I, II, and III [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. In contrast, stimuliation by carbachol induced significant phosphorylation of only protein I. This phosphorylation was low compared to isoproterenol-induced phosphorylation but corresponded to the smaller enhancement of amylase secretion. The mouse organ, however, is almost equally sensitive to beta-adrenergic and to cholinergic agonists. Incubation of mouse parotid gland slices with either 20 microM isoproterenol or 10 microM carbachol resulted in strong and comparable releases of amylase, which were accompanied by comparable phosphorylations of protein I. Proteins II and III were phosphorylated only in the presence of isoproterenol. Removal of external calcium by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate abolished the carbachol-induced release of amylase but not the phosphorylation of protein I. Isoproterenol-induced secretion of amylase and phosphorylation of proteins I, II, and III were not inhibited under these conditions. Amylase release stimulated by the ionophore A-23187 was accompanied by the phosphorylation of protein I. Two-dimensional electrophoresis revealed that the radioactive spot corresponding to protein I was located at the same position after cholinergic and after beta-adrenergic stimulation, indicating that both stimuli led to the phosphorylation of the same membrane-associated protein. These findings strongly support the view that the phosphorylation of protein I is an important step in the sequence of events leading from receptor activation to exocytosis.

Dissociation between stimulant-evoked acinar membrane resistance change and amylase secretion in the mouse parotid gland.

1. Isolated segments of mouse parotid gland were superfused with a physiological saline solution. Membrane potential and input resistance were measured with intracellular micro-electrodes. Amylase secretion was monitored using an automated fluorometric assay. The parotid gland was stimulated by exposure to isoprenaline (10(-6)M), ACh (10(-6) M), dibutyryl or monobutyryl cyclic AMP (10(-3)M). 2. ACh evoked a sharp decrease in input resistance (from about 3-6 M omega to 1-2 M omega ) accompanied by a modest (two-fold) increase in amylase output. Both effects were fully reversible. 3. Isoprenaline evoked a marked increase in amylase output (five to ten-fold) reaching maximum after 20-30 min of stimulation. Throughout the period of intensive secretion the input resistance remained at the control level. Short pulses of ACh stimulation dramatically reduced input resistance in this period. 4. Dibutyryl cyclic AMP and monobutyryl cyclic AMP caused marked increases in amylase output (five to ten-fold) peaking 40-50 min after start of the continuous stimulation. This increase in amylase secretion was not accompanied by any change in input resistance. Short pulses of ACh stimulation sharply and reversibly reduced input resistance both in control and test periods. 5. It is concluded that the ACh-evoked reduction in input resistance is not a consequence of the secretory event and that secretion does not itself cause a decrease in input resistance.

Substance P: indirect and direct effects on parotid acinar cell membrane potential.

Direct effects of exogenously applied substance P on salivary acinar cells have been previously reported. This electro-physiological study confirms these direct effects in rat but not mouse parotid gland. We demonstrate that in the absence of autonomic blockade the peptide evokes marked responses which are blocked by atropine (10(-6) M). These effects cannot be attributed to direct activation of acinar cells and are presumably due to release of acetylcholine from parasympathetic nerve endings. We consider that substance P, in addition to direct effects, could act to modulate neuronal activity in salivary glands; a role already assigned to the peptide in the central nervous system.

Electrophysiology of mouse parotid acini: effects of electrical field stimulation and ionophoresis of neurotransmitters.

1. Intracellular micro-electrode recordings of membrane potential and input resistance were made from surface acini of mouse parotid glands placed in a Perspex tissue bath through which oxygenated physiological saline solutions were circulated. The acinar cells were stimulated by microionophoresis of both acetylcholine (ACh) and adrenaline (Ad) from extracellular micropipettes, and by electrical field stimulation via a pair of platinum electrodes. 2. The acinar cells had a mean resting membrane potential of -64.9 mV +/- 0.6 S.E. The input resistance of the unstimulated cell was 4.63 M omega +/- 0.19 S.E. In a number of cells spontaneous miniature depolarizations were observed, associated with synchronous reductions in input resistance. 3. The responses to ionophoresis of both ACh and Ad and the response to supra-maximal field stimulation were identical. Stimulation always evoked a marked decrease in input resistance associated with an initial potential change, generally followed by a delayed hyperpolarization during which the input resistance returned to normal. 4. Field-stimulation responses could be evoked to single shock (1-2 msec) and to low frequency (1-4 Hz) stimulation. The latency for this response was 245 msec +/- 12 S.E. 5. The field-stimulation response was shown to be susceptible to blockade of nerve conduction in sodium-free or tetrodotoxin- (TTX-) containing media; and to blockade of neurotransmitter release in calcium-free media. 6. The field-stimulation and ACh responses were recorded at different levels of membrane potential within the same cells by applying either hyperpolarizing or depolarizing direct current through the recording electrode. The membrane potential at which the initial potential change undergoes reversal, i.e. changes from a depolarization to a hyperpolarization, is known as the equilibrium or reversal potential, EFS and EACh respectively. The field-stimulation (FS) and ACh responses underwent simultaneous reversal at about -60 mV, i.e. EFS = EACh. Equilibrium potentials were also determined indirectly by analysis of the responses evoked by each stimulus in the manner described by Trautwein & Dudel (1958). Using this technique the equilibrium potentials of the responses to all three stimuli, field stimulation, ACh and Ad, were again about -60mV, i.e. EFS = EACh = EAd. 7. Both the field-stimulation and ACh responses were abolished by atrophine (10(-6) M) while the response to Ad persisted. Atropine also abolished all spontaneous activity. The alpha-adrenergic blocker phentolamine (10(-5) M) abolished the response to Ad but left the field-stimulation response unaffected. 8. Electrical field stimulation of isolated segments of salivary gland evoked release of endogenous neurotransmitter as a consequence of neural excitation. The technique of field stimulation thus makes it possible to investigate the functional innervation of a gland using the in vitro preparation. In the mouse parotid gland the field stimulus response was mediated by ACh released from parasympathetic nerve endings.

Mediation of β-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced 45Ca ++ efflux and 2) a dependence on the extracellular Na + concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on 45Ca ++ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release 45Ca ++. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na + was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the 45Ca ++ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na +. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na + followed by release of intracellularly stored calcium.

Effect of ethanol upon isoproterentol stimulation of growth and secretion in the mouse parotid gland

Isoproterenol (0.3 mmole/kg body wt.), when injected into the mouse intraperitoneally, increases the weight by 35% and stimulates DNA synthesis 30-fold in the parotid gland. The induction of both hypertrophy and hyperplasia is completely inhibited by ethanol at a dose of 200 mmole/kg body wt. but is almost unaffected by 60 mmole/kg. The full inhibiton of both growth parameters is observed when ethanol is administered up to 5 hr after isoproterenol. Partial inhibition is observed when ethanol is given as long as 15 hr after isoproterenol. It contrast ethanol did not alter the secretion of α-amylase in response to isoproterenol. Ethanol had no effect upon the rise in cyclic GMP level caused by isoproterenol but augmented the rise in cyclic GMP In agreement with these in vivo observations, low concentrations of ethanol activated adenylate cyclase in vitro , however guanylate cyclase activity was quite strongly inhibited. Although high levels of ethanol (300 mmole/kg) inhibited the induction of both ornithine decarboxylase and S-adenosylmethionine decarboxylase little inhibition was seen at 200 mmole/kg suggesting that the interference with polyamine metabolism is not the mechanism of the ethanol effect upon isoproterenol-induced parotid growth.

Calcium mediation of cholinergic-stimulated amylase release from mouse parotid gland

Amylase release from mouse parotid fragments was stimulated independently by cholinergic and beta-adrenergic agents. The cholinergic agonist, carbachol, significantly increased release of amylase only in Ca2+ containing medium whereas isoproterenol-stimulated amylase release was unaffected by Ca2+ removal. The ionophore, A23187, mimicked the effect of cholinergic stimulation when Ca2+ was present in the medium. Uptake of 45Ca2+ into tissue fragments was enhanced by carbachol and A23187 but not by isoproterenol; atropine blocked the effect of carbachol. Diphenylhydantoin (DPH) and verapamil partially inhibited carbachol-stimulated amylase release and 45Ca2+ uptake, whereas diazoxide potentiated these effects; in all cases there was good parallelism between 45Ca2+ uptake and amylase release. It was concluded that the primary step in the release of amylase from mouse parotid gland in response to cholinergic agents is an increased influx of Ca2+.

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.

Membrane potential and resistance changes induced in salivary gland acinar cells by microiontophoretic application of acetylcholine and adrenergic agonists.

The effects of microiontophoretic applications of catecholamines and acetylcholine on parotid acinar cell membrane potential and resistance were investigated using intracellular microelectrode recording in superfused segments of mouse parotid or rat submandibular glands. Short pulses of acetylcholine and alpha-adrenergic agonists had similar effects, consisting of a marked decrease in membrane resistance accompanied by an initial depolization or hyperpolarization depending on the level of the resting membrane potential. This initial response was followed by a slow hyperpolarization occurring at a time when the resistance was increasing towards the prestimulation level. The equilibrium potential for the initial potential change caused by excitation of the cholinergic receptors was investigated directly by setting the membrane potential at different levels by injecting direct current and stimulating the same cell repeatedly with equal doses of acetylcholine. The equilibrium potential was found to be about -55 mV. The delayed hyperpolarization could not be reversed by passing hyperpolarizing current, but actually increased in size with higher membrane potentials. The minimum latency of the effect of acetylcholine or alpha-adrenergic agonists was 200-500 msec. Excitation of beta-adrenoceptors caused, after a long latency of several seconds, a small depolarization. Epinephrine induced a combined alpha- and beta-adrenergic response, with the alpha-component predominating. Blocking the alpha-adrenoceptors with phentolamine revealed the beta-adrenergic depolarization, while blocking the beta-adrenoceptors with propranolol caused the components of the alpha-adrenergic response to become more pronounced. All three receptors (alpha- and beta-adrenoceptors and cholinergic receptors) were present in individual acini.