Rat Parotid Salivary Glands Research Articles

Influence of parasympathetic non-adrenergic, non-cholinergic mechanisms on [ 3H]thymidine uptake in rat parotid salivary glands in response to mastication following upon a liquid regimen

A role for parasympathetic non-adrenergic, non-cholinergic (NANC) mechanisms in parotid gland cell hyperplasia in response to mastication was investigated. Following a liquid regimen for 1 week, aiming at glandular inactivity, rats were offered hard chow for 2 h to throw their parotid glands into secretory activity. In response to the increasing demands, [ 3H]thymidine incorporation into the trichloroacetic acid-insoluble material increased by 76% in normally innervated glands, while there was no increase in glands parasympathetically denervated in advance. In innervated glands of rats pretreated with atropine and α- and β-adrenoceptor blockers the increase was 46%. The results suggest a potential contribution of parasympathetic NANC mechanisms to the mitotic response of the parotid gland under natural feeding conditions.

Adrenoceptor-activated nitric oxide synthesis in salivary acinar cells.

We investigated the cellular regulation of nitric oxide synthase (NOS) activity in isolated acinar cells from rat parotid and human labial salivary glands, using the newly developed fluorescent nitric oxide (NO) indicator, DAF-2. We found that sympathetic stimulation with norepinephrine (NE) caused a strong increase in NO synthesis that was not seen after parasympathetic stimulation with acetylcholine. In rat parotid acinar cells, we furthermore investigated to which extent the NOS activity was dependent on the intracellular free Ca2+ concentration ([Ca2+]i) by simultaneously measuring NO synthesis and [Ca2+]i. It was found that a simple correlation between the rise in [Ca2+]i and the rate of NO production following NE stimulation does not exist, and studies in which [Ca2+]i was elevated by means of the Ca2+ ionophore, ionomycin, further established that even a very large rise in [Ca2+]i did not cause significant NO synthesis. We furthermore found that activating adrenoceptors with NE causes synthesis of cGMP by activating a guanylyl cyclase, and that an enhanced [cGMP] evoked by use of caged cGMP causes Ca2+ release from internal stores. Thus, upon sympathetic stimulation, salivary gland acini synthesize NO that, in addition to playing a role in controlling intracellular [Ca2+]i, also might play a role in retrograde signaling processes to the surrounding tissue.

Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.

Several members of the Na+/H+ exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+ exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+ exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50 = 0.014 +/- 0.005 microM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+ exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50 = 0.754 +/- 0.104 microM) than cell lines expressing NHE1 or NHE2 (IC50 = 0.076 +/- 0.013 or 0.055 +/- 0.015 microM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50 = 6.25 +/- 1.89 microM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+ exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.

Development and characterization of SV40 immortalized rat parotid acinar cell lines.

Rat parotid salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E1, and forskolin were effective activators of intracellular cyclic adenosine 3':5'-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.

Cell deletion by apoptosis during regression of rat parotid sialadenosis.

Enlargement of the rat parotid salivary glands was induced by repeated administration of isoproterenol. Mean wet weights of the treated glands increased steadily to 240% of control values. Following withdrawal of the drug, quantitative histological techniques were used to investigate the balance between hypertrophy, hyperplasia and apoptosis. The volume occupied by acinar cells relative to the total gland volume together with cytoplasmic magnitude of nuclear area ratios as measures of hypertrophy increased during the early experimental period. Similarly, serous acinar cell mitotic counts increased, indicating that hyperplasia had occurred. Apoptosis was demonstrated at light microscopical level to be the main mechanism for cell deletion as the glands returned to normal size and weight. The results indicate that hypertrophy and hyperplasia of serous acinar cells contribute to isoproterenol-induced sialadenosis. The experimental animal model demonstrates that these proliferative changes are completed by 48 h and thereafter are balanced by apoptosis as the glands recover their normal size and weight.

Influence of atrial natriuretic peptide on cyclic nucleotides and amylase release in rat parotid salivary gland in vitro

Atrial natriuretic peptide (ANP), sodium nitroprusside and hydroxylamine increased cGMP accumulation in rat parotid acinar cells both in the presence and absence of forskolin but in a different manner. On the other hand, ANP decreased forskolin-stimulated cAMP accumulation, although sodium nitroprusside and hydroxylamine had no effect on cAMP accumulation. Amylase release stimulated by forskolin, dibutyryl-cAMP or isoproterenol was depressed by ANP, whereas sodium nitroprusside and hydroxylamine did not evoke the inhibition of forskolin-stimulated amylase release. These results suggest that the inhibition of cAMP accumulation and of amylase release by ANP were not mediated via cGMP produced by guanylate cyclase-A.

Reciprocal expression of c-jun, proline-rich protein and amylase genes during rat parotid salivary gland development

Abstract We have investigated the temporal expression and cellular localization of the c-jun proto-oncogene and two major rat parotid gland secretory protein genes, PRP (proline-rich protein) and amylase, during postnatal development. c-jun mRNA steady-state levels increased at days 1, 7 and 14 after birth and decreased to basal levels at 21 days and older. PRP mRNA was first detected at 14 days and abruptly increased to adult levels at day 21. Amylase transcripts were first seen at day 7 and progressively increased to adult levels by 28 days. In situ hybridization demonstrated c-jun mRNA accumulation in the differentiating acinar cells and the ducts. The c-jun mRNA accumulation with time corresponds with the proliferative activity reported to occur in these two cellular populations. PRP transcripts were present exclusively in the well differentiated acinar cells while the accumulation of amylase mRNA corresponded to the progressive commitment of parotid cells to acinar differentiation. Our data suggest that during the postnatal development of the rat parotid gland: (a) c-jun expression associates with parotid gland proliferation and precedes the expression of PRP and amylase genes, and (b) activation of PRP and amylase genes is not concomitant and apparently occurs only in differentiating acinar cells.

Salivary insulin-like growth factor-I originates from local synthesis.

Insulin-like growth factor-I (IGF-I) is a GH-dependent growth factor found in its highest concentrations in plasma. It is also measurable in saliva. The origins of salivary IGF-I concentrations were studied. Intracardial administration of Sprague-Dawley rats with 125I-labelled IGF-I and subsequent analysis of plasma and saliva samples by exclusion gel chromatography and SDS-PAGE, followed by autoradiography, demonstrated the apparent inability of IGF-I to cross from the plasma pool through to saliva. 125I-Labelled IGF-I was not chromatographed immediately before injection, resulting in administration of free iodide along with the iodinated peptide. This free iodide was demonstrable in saliva, indicating that movement of substances from plasma to saliva was measurable using the levels of 125I activity administered. Free iodide in saliva was not contributed to by 125I-labelled IGF-I degradation since 125I-labelled IGF-I was shown to be stable in saliva over 24 h. These data indicated that IGF-I in saliva is produced locally. Identification of a 4.7 kb IGF-I mRNA transcript in rat parotid salivary gland was consistent with IGF-I synthesis within that tissue.

The effects of moderate protein deficiency on β-adrenoceptor density in rat parotid and submandibular salivary glands

The effects of moderate protein deficiency on the number and dissociation constant of β-adrenergic receptors were investigated. One group of adult rats was fed a protein-deficient diet (5% protein), a control group was given a similar diet with 20% protein, and a third group was fed a standard pellet diet. After 21 days, the salivary glands were removed and the cell membranes were isolated. The binding of [ 3H]-dihydroalprenolol to cell membranes was measured, with or without the presence of timolol, a potent β-adrenoceptor antagonist. The rats fed the 5% protein diet had fewer β-adrenoceptors in both the parotid and the submandibular glands than did the 20% protein group, which did not differ from the standard pellet group. The dissociation constant was not affected by protein deficiency. The rats fed the 5% protein diet had a significantly lower gain in body weight, less heavy submandibular glands but heavier parotids than the rats fed 20% protein. Thus the cellular conditions for protein secretion in the salivary glands are impaired during moderate protein deficiency in the adult rat.

Progesterone metabolism by major salivary glands of rat—II. Parotid gland

The metabolism of [4- 14C]progesterone in the parotid salivary glands of nonpregnant female, pregnant female and male rats was investigated in vitro. The metabolic activity of the male rats was significantly lower than in either of the female groups. The pregnant group was metabolically more active than the nonpregnant female group, but this difference was not statistically significant. In homogenates and soluble fractions the main metabolite was 20α-hydroxy-4-pregnen-3-one in female rats. In male rats the main metabolites were 20α-hydroxy-4-pregnen-3-one and 3α-hydroxy-5α-pregnan-20-one in homogenates and 20α-hydroxy-4-pregnen-3-one in soluble fractions. In the microsomal fractions of both sexes polar compounds predominated. The results indicated the presence of at least the following progesterone metabolizing enzymes in rat parotid salivary glands: 3α-, 3β-, 20α- and 20β-hydroxysteroid dehydrogenase, 5α- and 5β-steroid hydrogenase and 17α-steroid hydroxylase activities. In the homogenates and soluble fractions of female rats 20α-hydroxysteroid dehydrogenase activity was significantly higher than in males.

Two novel c- abl mRNAs are expressed in rat parotid salivary glands during in vivo β-adrenergic receptor stimulation

The c- abl proto-oncogene is transcribed in most cell lines and tissues into two mRNAs of 6.5 and 5.3 kb, which have different 5′ ends and encode two 150 kDa proteins that are largely colinear, but have different N-termini. We show here that two unusually short and abundant c- abl-related mRNAs of 1.5 and 1.3 kb appear in rat parotid salivary glands, within 1 day of in vivo administration of the β-adrenergic receptor agonist isoproterenol. These transcripts are not found in the submandibular salivary gland or in the heart and they are too short to encode the known c- abl proteins. RNA blot, S1 nuclease protection and primer extension analysis suggest that the isoproterenol inducible parotid gland mRNAs do not contain the kinase domain, but represent part of the C-terminal segment of the abl reading frame.

Morphometric study of the microvasculature of the main excretory duct subepithelia of the rat parotid, submandibular and sublingual salivary glands

This study investigated the relationship between the fine structure of the main excretory duct (MED) epithelia and the subepithelial capillaries of the major salivary glands (parotid, submandibular, and sublingual) in the rat. The MED subepithelial capillaries vary in type (fenestrated and continuous), number of fenestrae present in the available fenestrated endothelium (the attenuated area of endothelium), and number of capillaries per unit length of epithelium. In the parotid gland, continuous capillaries predominate, and the capillary density per 200 microns of epithelium (1.73 in region I and 1.03 in region II) is lower than in the other two glands, as is the number of fenestrae per 10 microns of available endothelium (1.97 in region I and 1.25 in region II). The submandibular gland has mainly fenestrated capillaries. The capillary density per 200 microns of epithelium (4.46 in region I and 5.51 in region II) is higher than in the other two glands, as is the number of fenestrae per 10 microns of available endothelium (4.82 in region I and 7.90 in region II). Similarly, in the sublingual gland, fenestrated capillaries are dominant. The density of capillaries per 200 microns of epithelium (4.15 in region I and 4.31 in region II) is intermediate between the parotid and submandibular glands, as is the number of fenestrae per 10 microns of available endothelium (2.98 in region I and 3.83 in region II).

Changes in microfilament distribution during amylase exocytosis in rat parotid salivary glands in vitro

In parotid acini, microfilaments were predominantly localized at the luminal border when visualized with the fluorescent probe, rhodamine-phalloidin. There was no fluorescence in the cytoplasm. When acini were stimulated by isoprenaline, the membranes of the secretory granules appeared to become undercoated by microfilaments immediately after fusion with the luminal membrane. The intensity of fluorescence was correlated with the extent of enlargement of the lumina. These findings indicate that the microfilament system (actin-myosin contractile system) may play a role in regulating the movement of the luminal plasma membrane rather than in the control of transport and access of secretory granules to the lumen.

Influence of β-adrenergic stimulation on glycosylation of a major, secretory N-linked glycoprotein from rat parotid salivary gland

β-Adrenergic stimulation with 10 μM isoproterenol increased the [ 3H]-mannose/[ 14C]-leucine ratio (three- to six-fold) of protein extracts in double-radiolabelled rat parotid acinar cells. Characteristics of oligosaccharides in a major parotid glycoprotein ( M r ~ 220,000; gp 220) were studied. Gp 220 from control and experimental cells was endoglycosidase H-insensitive, endoglycosidase F-sensitive and bound both concanavalin A and wheat germ agglutinin. Gp 220 was removed from concanavalin A-Sepharose by sequential elution with 10mM α-methyl glucoside and 0.5 M α-methyl mannoside. These findings suggest that (1) oligosaccharides in gp 220 have both a biantennary complex and hybrid oligosaccharide chains, and (2) β-adrenoreceptor stimulation has little effect on the gross oligosaccharide structures of this glycoprotein.

The influence of varying the electrical frequency of sympathetic nerve stimulation on fluid and calcium secretion of the rat parotid salivary gland

The effects of varying this frequency were studied in the absence and presence of the specific adrenergic antagonists. There were no appreciable differences in fluid and calcium secretion at different frequencies of stimulation when the nerve was stimulated alone or in the presence of the selective antagonists. Thus, it is difficult to differentiate between the effects of α- and β-adrenergic receptor stimulation by varying the frequency of nerve stimulation. The findings also confirmed that both types of receptors are activated together when the sympathetic nerve is stimulated, β-Adrenergic receptors play a major role in secretion of both fluid and Ca; α-adrenergic receptors seem to have a minor role in their regulation.

Protein synthesis and amylase messenger RNA content in rat parotid salivary glands after total or partial stimulation with isoproterenol

The rate of synthesis of secretory proteins increases significantly in rat parotid glands after stimulated discharge of stored proteins. How any difference in the amount of secretory protein discharge affects the rate of subsequent protein synthesis, and whether this post-secretory synthesis is regulated at the level of messenger RNA, was now examined. One group of rats was stimulated to secrete 97% of stored secretory proteins by an intraperitoneal injection of isoproterenol. The other group received a much smaller dose to induce the discharge of about 40% of the proteins. Despite this difference in secretion, the subsequent rates of total protein synthesis, as well as of amylase, were increased to about the same extent. The amylase messenger RNA (mRNA) was identified and quantified by hybridization with a 32P-labelled amylase complementary DNA (cDNA) probe. The amylase mRNA in stimulated and unstimulated rats was of the same molecular size (Northern blot analyses). The amount of amylase mRNA, determined by dot blot analyses, were also increased in stimulated rats, although this increase was not as great as that in the rate of amylase protein synthesis. The implications of this discrepancy concern the possibility that the mechanism of regulation of secretory protein synthesis in parotid glands is at the translational level.

Secretion of amylase from the rat parotid salivary gland after degeneration of the auriculotemporal nerve

The output of amylase into saliva secreted after injection of methacholine or substance P was increased after parasympathetic denervation, but the salivary concentration of amylase was unchanged. The increased output corresponded to the increased flow. Isoprenaline injected during the methacholineinduced secretion raised the output, more being secreted from the denervated than from the contralateral gland. Vasoactive intestinal peptide, given while substance P caused salivation, also increased the amylase output, but equally from the two glands.

Effect of cytochalasin D on acinar cell structure and secretion in rat parotid salivary glands in vitro

Cytochalasin D, a microfilament disrupting agent, considerably inhibited isoproterenol-stimulated amylase release from enzymatically dispersed parotid acini. Histologically cytochalasin D caused a loss of microvilli lining acinar lumina and luminal enlargement. Nearly empty vacuoles appeared near the luminal and lateral surface, and the membrane bordering on the vacuoles was often continuous with the plasma membrane. Therefore, the vacuolization probably resulted from an elongation of the membrane lining the lumen. Fluorescence staining with rhodamine-phalloidin showed that cytochalasin D caused disruption of microfilaments. When stimulating the cytochalasin D-treated cells with isoproterenol, the number of secretory granules in the cytoplasm diminished markedly and secretory material was observed in the vacuoles, indicating that inhibition of amylase release by cytochalasin D is not due to blocking of exocytosis but to the retention of amylase discharged into vacuoles. These findings suggest that microfilaments are essential in maintaining the parotid acinar structure but do not play a direct part in the movement of secretory granules and their fusion with the luminal membrane.

Restoration of α-Lactalbumin-inhibited rat parotid salivary gland hypertrophy and hyperplasia by agents specific for membrane glycoprotein N-acetylglucosamine

Chronic isoproterenol treatment of rats results in hypertrophy and hyperplasia of the parotid gland. This physiological change appears to be mediated in part by cell surface 4β-galactosyltransferase activity (EC No. 2.4.1.38) as the specific modifier protein. α-Lactalbumin, when injected concomitantly with isoproterenol, prevented both gland hypertrophy and hyperplasia. The further incorporation of agents with specificity for terminal N-acetylglucosamine residues of membrane glycoproteins in the above drug regimen resulted in the restoration of parotid gland hypertrophy and hyperplasia. These agents included soluble bovine 4β-galactosyltransferase and the lectin wheat-germ agglutinin. When plasma membranes were isolated from isoproterenol-treated parotid gland acinar cells, a new membrane glycoprotein with an apparent molecular mass of 30,000 Da was identified. This protein was not present in the membranes from control parotid glands. Therefore the transition from quiescent to active parotid acinar-cell proliferation appears to require two membrane events; the appearance of cell-surface galactosyltransferase and a new membrane glycoprotein substrate.

Inducing effects of chronic administration of isoproterenol on 1-acyl-sn-glycero-3-phosphate and 1-acyl-sn -glycero-3-phosphocholine acyltransferases in rat parotid salivary gland

1. 1. In rat parotid gland, chronic administration of isoproterenol caused significant increase of linoleic acid and decrease of arachidonic acid at the sn-2 position of phosphatidylcholine. 2. 2. The activities of 1-acyl-sn-glycero-3-phosphate and 1-acyl-sn-glycero-3-phosphocholine acyl-transferases were increased 3–8-fold and 2-fold, respectively, in the parotid gland microsomes of isoproterenol-treated rat. 3. 3. Furthermore, the specificity of these two enzymes for various acyl-CoAs was also changed by administration of isoproterenol. 4. 4. The alteration of unsaturated fatty acid composition at the sn-2 position of phosphatidylcholine was at least in part due to the change of activity and substrate specificity of lysophospholipid acyltransferases.