Regulation of cytosolic Ca2+ in resting and stimulated rat submandibular salivary gland acini

To determine the range over which cytosolic Ca2+ concentration (Cai2+) is regulated, Cai2+ was measured in salivary acini loaded with Fura-2. With extracellular Ca2+ (Cao2+) = 2.5 mM, mean resting Cai2+ (+/- SD) was 103 +/- 20 nM (n = 29). Carbachol increased Cai2+ immediately to 288 +/- 81 nM, which then declined to a sustained level of 253 +/- 58 nM. When Cao2+ was less than 10 nM, the immediate response to carbachol was little affected but the sustained response was abolished. Atropine abolished all responses to carbachol. Changes in Cai2+ were not due to Ca2+-induced Ca2+ release or to Ca2+ influx via voltage-dependent channels. With Cao2+ = 2.5 mM, epinephrine raised Cai2+ initially to 196 +/- 57 nM (n = 6), which soon declined to stable levels of 178 +/- 40 nM. When Cao2+ was less than 10 nM the prolonged response to epinephrine was abolished but the initial increase was relatively unaffected. All epinephrine-induced changes in Cai2+ were abolished by prazosin. The range of Cai2+ measured corresponds to that over which non-mitochondrial Ca2+ transport is stimulated in permeabilized submandibular gland acini suggesting that such a Ca2+ transport system may regulate physiological changes in Cai2+ in these acini in the rat.     

Abnormal 45Ca fluxes in dispersed submandibular acini of rats treated with reserpine

The uptake and efflux of the isotopic tracer 45Ca were compared in dispersed submandibular acini of both control rats and rats treated with seven daily doses of reserpine (0.5 mg/kg, i.p.). Tracer uptake occurred in a time-dependent manner in both types of acini and reached 8.4 +/- 0.2 and 8.0 +/- 0.2 pmol/mg protein, respectively, in acini from control and treated animals after 60 min of incubation. Uptake of tracer was 2.35 nmol/mg DNA in control cells and 4 nmol/mg DNA in cells from treated rats at 60 min. 45Ca uptake (per mg protein) was enhanced in control acini 48% by 20 mumol/L epinephrine; 38% by 50 mumol/L carbachol; and 23% by 10 mumol/L isoproterenol. A similar order of potency was observed when uptake was expressed per mg DNA. In acini from reserpine-treated rats, 45Ca uptake (per mg protein) was increased 53% by epinephrine, 39% by isoproterenol, and only 8% by carbachol. The same enhanced effect of isoproterenol and lack of effect of carbachol were observed when uptake was calculated per mg DNA. In the absence of secretagogue, efflux of 45Ca from tracer-pre-loaded acini was larger in acini from reserpine-treated rats (53%) than in control acini (36%). Whether expressed in terms of mg protein or mg DNA, this efflux was increased in control acini 35% by epinephrine, from 25 to 28% by isoproterenol, and 17% by carbachol. In acini of reserpine-treated rats, epinephrine increased 45Ca efflux 20%, isoproterenol from 25 to 28%, and carbachol from 14 to 15%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of alpha-receptor stimulation on Cl transport by rat submandibular acini

Dispersed salivary acini isolated from the rat submandibular gland by enzymatic digestion were used to study the effects of alpha-receptor stimulation on transmembrane transport of 36Cl. In the absence of secretagogue, the tracer accumulated in the cells in a time-dependent manner until a steady-state content of 6.8 +/- 0.1 nmol/mg protein was attained after 3-5 min of incubation. Epinephrine (1 mumol/L) alone did not modify 36Cl accumulation but in the presence of the beta-receptor blocker propranolol (1 mumol/L) caused a significant (21%) reduction in the isotope content of the cells to 5.2 +/- 0.1 nmol/mg protein. In acini pre-loaded with 36Cl for 12 min, 1 mumol/L epinephrine caused a rapid but transient net efflux of tracer, but the isotope content subsequently increased to pre-stimulation levels. In the presence of propranolol, however, the efflux of 36Cl induced by epinephrine was larger and more sustained and was partially inhibited by the K-channel blocker quinidine (1 mmol/L) and significantly by the absence of Ca2+ in the incubation medium. The alpha-agonist phenylephrine (10 mumol/L) also significantly reduced the steady-state 36Cl content of tracer-pre-loaded cells. By contrast, exposure of the acini to epinephrine in the presence of the alpha-receptor blocker phentolamine, or the beta-agonist isoproterenol, increased the tracer content of the cells, whether the drugs were added at time zero or to tracer-pre-loaded cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium dependency of adrenergic and muscarinic cholinergic stimulation of mucin release from dog submandibular gland cells.

Stimulation of muscarinic cholinergic, alpha-adrenergic and beta-adrenergic receptors elicited mucin release from dispersed dog submandibular cells. The secretory response to acetylcholine was much more pronounced than to adrenergic agonists, and largely dependent on the presence of extracellular Ca2+, but the dependency on extracellular Na+ was slight. Ionomycin also stimulated mucin release. In rat submandibular cells, neither muscarinic cholinergic agonists nor ionomycin were as effective mucosecretagogues as beta-adrenergic agonists. alpha-Adrenoceptor-mediated release was decreased by chelating extracellular Ca2+ with EGTA. The beta-adrenoceptor-mediated response was diminished by extensive exposure of cells to EGTA, due at least in part to the requirement of Ca2+ for beta-adrenoceptor stimulation of cAMP formation. 8-br-cAMP stimulated 45Ca2+ release from cells preloaded with 45Ca2+. The 8-br-cAMP-induced mucin release was eliminated in ionomycin-pretreated cells, but not inhibited by chelating extracellular Ca2+ and by the treatment of the cells with TMB-8 or in the cells loaded with BAPTA. These results suggest that not only the adrenergic system but also the muscarinic cholinergic system may participate in the regulation of mucin release in dog submandibular gland, and also provide the possibility that, in addition to a cAMP-mediated mechanism, Ca(2+)-dependent mechanisms may be involved in mucosecretion in dog submandibular acini.     

The control of cytosolic Ca2+ concentration: studies of high affinity Ca2+ transport in permeabilized acini of rat submandibular glands

Active Ca2+ transport was studied in acini that had been permeabilized by incubation in buffer nominally free of Ca2+ in order to avoid any contribution to measured Ca2+ uptake by elements of the plasma membrane. In most experiments, ruthenium red was used to inhibit mitochondrial Ca2+ uptake. Non-mitochondrial Ca2+ transport was greatest at pH 7.0-7.5 and required Mg2+-ATP at concentrations typical of the cytosol (K0.5 = 1.36 +/- 0.53 mM). Other substrates were much less effective than ATP. Ca2+ uptake was stimulated by Ca2+ at concentrations near those measured in intact cells (K0.5 = 0.43 +/- 0.17 microM). Hill coefficients at subsaturating concentrations of Ca2+ (1.08 +/- 0.27) and Mg2+-ATP (0.81 +/- 0.22) indicated that cooperative interactions are not characteristic of the major cationic regulators of Ca2+ transporting activity. Na+ did not release Ca2+ from acinar mitochondria, but consistently reduced non-mitochondrial Ca2+ uptake by about 20% as compared to uptake in the presence of an equimolar K+ concentration. The properties of non-mitochondrial Ca2+ uptake in permeabilized acini are similar to those of high affinity Ca2+ uptake which, in broken cell preparations, has been found distributed in parallel with elements of the endoplasmic reticulum. The Ca2+-sequestering properties of a non-mitochondrial organelle in permeabilized submandibular gland acini are those expected of a principal regulator of cytosolic Ca2+ and could account for the ionized Ca2+ concentration measured in resting salivary acinar cells.     

Physostigmine-induced salivary secretion in the rat

The purpose of this study was to see if physostigmine, a reversible cholinesterase inhibitor, affects the secretion and composition of saliva of the major salivary glands of the rat. Low doses of physostigmine did not elicit secretion. At higher doses there was significant flow from the parotid and submandibular glands within 5 min; however, no sublingual secretion was observed. The submandibular flow rate was highest for the first 5 min, then declined rapidly. The parotid flow rate initially was one-fifth of the maximum submandibular rate and then gradually decreased. The concentrations of Ca, Na and K of physostigmine-induced parotid saliva, and the Na of submandibular saliva, were similar to those with carbachol stimulation. The Ca and K concentrations of submandibular saliva were significantly higher than with carbachol or parasympathetic stimulation, and resembled those of alpha-adrenergic stimulation. The protein concentrations of physostigmine-evoked saliva from both glands were similar. The amylase activity of physostigmine-evoked parotid saliva was much higher than that of carbachol or parasympathetic stimulation. Physostigmine-evoked secretion was completely blocked by atropine, a cholinergic antagonist, and by reserpine, partially blocked by phentolamine, an alpha-adrenergic antagonist and not affected by surgical sympathectomy. Morphologically, physostigmine resulted in a moderate decrease in the number of acinar, but not ductal, secretory granules of both the parotid and submandibular glands, while the sublingual gland was unaffected. Numerous patches of parotid acini also developed vacuoles or vesicles. These results suggest that physostigmine-induced salivary secretion is mediated primarily by direct effects on cholinergic and alpha-adrenergic receptors.     

Degranulation of acinar cells in von Ebner's gland of the rat.

We investigated the effect of sympathetic agonists, parasympathetic muscarinic agonists and substance P on depletion of secretory granules in acinar cells of rat von Ebner's gland. Drugs were injected intraperitoneally at several different concentrations. Antagonists were given 15 minutes before injection of the agonist, and the extent of depletion of secretory granules in glandular acini was calculated using a computerized color image analyzer. The specific alpha 2-sympathetic agonist clonidine and the beta 1-sympathetic agonist dobutamine produced a depletion of secretory granules. When combined with injections of the alpha 2-sympathetic antagonist yohimbine and the beta 1-sympathetic antagonist acebutolol, depletion of secretory granules was blocked. The parasympathetic muscarinic agonist carbachol also produced a depletion of secretory granules. QNB blocked the depletion caused by carbachol, while atropine partially inhibited depletion. The specific M1-muscarinic agonist McN-A-343 caused some depletion, although there was no significant differences between it and the control. Complete depletion of the secretory granules was achieved by carbachol stimulation superimposed on substance P stimulation. We concluded that the activation of the sympathetic alpha 2- and beta 1-receptors, as well as the M2 (M2 beta)-muscarinic and substance P receptors, results in degranulation of acinar cells in von Ebner's gland of the rat.     

The influence of vasoactive intestinal peptide receptors in dispersed acini from rat submandibular gland on cyclic AMP production and mucin release

Vasoactive intestinal peptide has been identified as an important regulator of submandibular salivary gland function, consistent with its localization with acetylcholine in parasympathetic neurones innervating this gland. Enzymatically dispersed acini from rat submandibular gland are a useful system in which to study gland regulation at the cellular level. Here, three aspects of vasoactive intestinal peptide interactions with acini were examined: inhibition of binding of [125I]-vasoactive intestinal peptide, stimulation of cyclic AMP production and enhancement of mucin release. Vasoactive intestinal peptide and peptide histidineisoleucineamide inhibited [125I]-vasoactive intestinal peptide binding to intact acini, with IC50 values of 16 +/- 3 and 46 +/- 17 nM, respectively. This rank order of potency agrees with that observed previously in assays using rat submandibular gland membranes and is similar to values obtained in assays measuring increases in cyclic AMP production in which the ED50 values for vasoactive intestinal peptide and peptide histidineisoleucineamide were 3.1 +/- 1.8 and 29 +/- 13 nM, respectively. Although vasoactive intestinal peptide stimulation of cyclic AMP production was only about 10% of that seen in response to isoproterenol, the levels of mucin release induced by the two agents were more similar. The ED50 for vasoactive intestinal peptide-stimulated mucin release was 0.12 +/- 0.05 nM, thus suggesting an activation anomaly in the vasoactive intestinal peptide receptor-coupled signal transduction pathway at a point between cyclic AMP production and mucin release.     

Postnatal changes in calcium and amylase of rat salivary glands, including calcium changes with senescence

Postnatal changes occur in glandular Ca concentration of rat parotid and submandibular glands. At 4 days of age, Ca concentration was low in both glands (only one-third to one-half that of adults) and increased gradually with age. The pattern of change was generally similar for male and female rats, but in submandibular gland, adult levels of 9-10 m-equiv./kg were reached by weaning, whereas for parotid gland, a gradual increase in Ca concentration occurred with adult levels of 9-10 m-equiv./kg reached by 7 weeks of age. The pattern of change was the same whether Ca concentration was expressed per kg wet or dry weight albeit water content changed with age. The changes in Ca concentration of parotid paralleled the age-associated increases in amylase activity of parotid gland. Amylase activity of submandibular gland was much less than that of parotid and similarly low at all ages examined, and did not parallel the age-associated increases in Ca concentration. The regulatory role of the sympathetic innervation on glandular Ca concentration was examined by effecting surgical denervation of parotid and submandibular glands at 8 days of age, and then determining Ca concentration of the denervated glands at 32 days. A three-fold increase in Ca concentration, similar to that following acute sympathectomy in adults, occurred in submandibular gland but no change was seen in parotid. An unexpectedly high concentration of Ca was also found in submandibular (but not parotid) gland of old rats.     

Effects of forksolin, dibutyryl cAMP and H89 on Ca2+ mobilization in submandibular salivary cells of newborn rats.

The effects of substances which affect cAMP or the cAMP-dependent protein kinase (PKA) on the inositol 1,4,5-trisphosphate (IP3) and Ca2+ responses to acetylcholine or thapsigargin were investigated in submandibular gland cells of newborn rats. Exposure to forskolin, dibutyryl cAMP or the PKA inhibitor H89 did not affect the formation of IP3 or the release of Ca2+ from intracellular stores elicited by acetylcholine. However, the thapsigargin-induced Ca2+ release was reduced by dibutyryl cAMP and enhanced by H89 in immature cells. Ca2+ influx activated by acetylcholine and thapsigargin was additive in immature cells but not in mature cells, suggesting the presence of a separate Ca2+ entry pathway in immature cells. Moreover, the acetylcholine-stimulated Ca2+ influx was significantly potentiated by forskolin and dibutyrylcAMP, but not by H89 in immature cells. In contrast, the thapsigargin-activated Ca2+ influx was dramatically enhanced by H89, but not by forskolin and dibutyrylcAMP in these cells. This modulation of Ca2+ mobilization by the test substances is different from that observed in mature submandibular cells in which forskolin, dibutyrylcAMP and H89 affected both IP3 formation and Ca2+ release in response to acetylcholine. Therefore, these results suggest differences in the interaction between the cAMP-PKA and the phosphoinositide-Ca2+ signalling pathways of mature and immature salivary cells. The modulation of Ca2+ influx by the cAMP-PKA pathway in immature cells is likely to play a part in the maturation of salivary cells.     

The identification of the sympathetic neurons innervating the hamster submandibular gland and their electrophysiological membrane properties

The neuron innervating the hamster submandibular (SM) gland was identified in the superior cervical ganglion (SCG) in vitro by recording the antidromic response using the intracellular recording technique. After the cellular response was recorded, methylene blue was injected iontophoretically into the neuron from the recording electrode, and the location of the cell soma was determined. The salivatory neurons of the SM gland were in the small- to medium-sized group of the entire cell population of the SCG. The cell size was 36.3 x 24.4 microm (mean, n=45). The postganglionic fibers were entirely unmyelinated (mean: 0.34 m/sec at 28-30 degrees C, n=141). Eighty-seven percent of the cells were distributed in the central one-third of area between the external carotid nerve origin and the caudal pole in the SCG. The resting membrane potential, membrane input resistance, membrane time constant and membrane input capacitance of the salivatory neuron were as follows: -49.2+/-7.6 mV (n=102), 52.9+/-23.6 Mohms (n=71), 8.0+/-3.4 msec (n=71) and 147+/-50 pF (n=71). Fast- and slow-excitatory postsynaptic potentials (EPSPs) were evoked, but not slow-inhibitory postsynaptic potentials (IPSPs). The fast EPSP was 13.1+/-5.7 mV in amplitude and 46.2+/-17.1 msec in duration (n=35). The slow EPSP (20 Hz, 5 sec) was 6.9+/-11 .9 mV in amplitude and 101+/-43 sec in duration (n=16). The directly-evoked spike was 63.0+/-11.9 mV in amplitude and 5.9+/-1.3 msec in duration (n=54). The spike after-hyperpolarization (AHP) was 12.5+/-3.5 mV in amplitude and 353+/-161 msec in duration. Na+ and Ca+ channels were involved in the spike generation. The voltage-dependent K+ channels (delayed rectifier), A channels and rapidly Ca2+-activated K+ channels (BK channels) regulated the spike-falling phase. The delayed rectifiers, A channels, and BK and SK (slowly Ca2+-activated) channels were involved in generation of spike-AHP. Muscarine suppressed the Ca2+ component of spike via muscarinic receptors.     

Effects of radiation on Ca2+ signaling in salivary epithelial cell lines transfected with Bcl-2 and Bcl-XL

The effects of radiation on the Ca2+ signaling system in HSY cells transfected with the Bcl-2 or Bcl-XL gene were studied. Bcl-2 overexpression did not alter carbachol (CCh)-elicited initial increase in cytosolic free Ca2+ concentrations ([Ca2+]i), but Bcl-XL overexpression dramatically reduced this response. Exposure to 10 Gy gamma-ray did not alter basal [Ca2+]i. By contrast, the CCh-stimulated initial [Ca2+]i increase was reduced at 0.5 and 4 h post-irradiation in all cell types and remained decreased at 24 h in wild-type and control-transfected cells, but recovered in Bcl-2- and Bcl-XL-transfectants. The formation of inositol 1,4,5-trisphosphate (IP3) in response to CCh at 4-h post-irradiation was decreased in wild-type and control-transfected cells, but not in Bcl-2 and Bcl-XL transfectants. The capacity of the IP3-sensitive Ca2+ store was significantly reduced by radiation in all cells except Bcl-XL transfectants. Ca2+ influx after stimulation with CCh was suppressed by exposure to radiation in wild-type and control-transfected cells, but not in Bcl-2- and Bcl-XL-transfectants. However, radiation enhanced Ca2+ influx activated by thapsigargin in all cell types. These results suggest that 1) radiation diminishes IP3 formation and Ca2+ release in response to CCh, but potentiates the store-operated Ca2+ influx; and 2) overexpression of Bcl-2 or Bcl-XL partially protects cells from radiation-induced inhibition of Ca2+ signaling.     

Alkalization produced by high-dose carbachol in HSG cell line is independent of Ca2+

OBJECTIVES: The aim of this investigation was to clarify the mechanism of alkalization induced by carbachol in HSG cells. MATERIALS AND METHODS: Cells of the HSG cell line derived from a human submandibular gland adenocarcinoma and those of the A-431 human epidermoid carcinoma cell line were loaded with a fluorescent pH indicator, BCECF/AM, and the change in the intracellular pH of adherent cells and suspended ones were measured following stimulation with various concentrations (10(-7) M to 10(-2) M) of neurotransmitters (carbachol, noradrenaline, and isoproterenol). RESULTS: Isoproterenol did not cause alkalization of either cell type, whereas, noradrenaline and carbachol alkalized both types over the concentration ranges of 10(-6) M to 3 x 10(-3) M (HSG cell by noradrenaline), 10(-7) M to 2 x 10(-4) M (A-431 cell by noradrenaline), and 7 x 10(-5) M to 10(-4) M (A-431 cell by carbachol). On the other hand, alkalization induced by carbachol in the HSG cells was recognized at concentrations higher than 6 x 10(-5) M, and it showed no upper limit in terms of carbachol concentration. This high-dose carbachol alkalization was not eliminated by preincubation with nifedipine (100 microM), a Ca2+ channel blocker, or with thapsigargin (100 microM), a microsomal Ca(2+)-ATPase inhibitor. CONCLUSIONS: The alkalization system induced by carbachol in the HSG cell was quite different from that in the A-431 cell, and that induced by high-dose carbachol in HSG cells appeared to be independent of intracellular Ca2+. These findings will be useful to clarify the mechanism of salivary secretion stimulated by neurotransmitters.     

颌下腺多形性腺瘤及其复发的临床研究

目的:评价颌下腺多形性腺瘤及其复发的临床特点及原因。方法:对8例颌下腺多形性腺瘤复发的临床特点和24例颌下腺手术野术中脱落细胞学进行分析及检测。结果:本院颌下腺多形性腺瘤术后有3例复发(3/366例,0.8%),另5例复发性颌下腺多形性腺瘤均为外院转入我院。8例病例复发时间均在1年以上,其中1例第2次复发时间为第1次复发术后5个月。1例复发性颌下腺多形性腺瘤术中见瘤体深叶处存留少许腺细胞,7例无腺细胞残留。术中脱落细胞显示8例原发性肿瘤手术野冲洗前有3例检测到腺泡细,5例为血液细胞,冲洗后2例为腺泡细胞,6例为血液细胞,8例复发性肿瘤冲洗前后均为1例腺泡细胞,7例血液细胞。8例非瘤性病变冲洗前1例为腺泡细胞,7例血液细胞,冲洗后均为血液细胞,3组之间无显著性差异(P>0.05)。结论:颌下腺多形性腺瘤复发罕见,可能与细胞学穿刺及瘤旁腺泡有关。     

MUC1 and the simple mucin-type antigens: Tn and Sialyl-Tn are differently expressed in salivary gland acini and ducts from the submandibular gland, the vestibular folds, and the soft palate

Objective and design

Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn.

Results

(1) The serous acini in the submucosal glands from the larynx and the soft palate expressed MUC1-associated glycans that were not detectable in the serous acini from the submandibular gland. (2) Virtually all the submucosal acini at oral site of the soft palate are mucous, and in contrast to mucous acini in the vestibular folds and submandibular gland, the palatinal acini in the submucosa underneath the oral mucosa showed a well-defined cytoplasmic reaction with anti-MUC1 antibodies as wells as with anti-Tn. (3) Both the mucous acini and the ducts at the oral site of the soft palate showed reaction for Sialyl-Tn while in the vestibular folds and in the submandibular gland expression for this carbohydrate was observed only in the acini. (4) The staining obtained after incubation with the Tn antibodies showed no cross localization with the staining obtained after incubation with an anti-A blood group antibody. (5) All the autopsies showed reaction in the glands after incubation with the MUC1 antibodies while some autopsies reacted with the anti-Tn antibodies and/or with the anti-Sialyl-Tn antibodies and others did not.

Conclusion

The mucin expression in the acini and ducts from the upper human aerodigestive tract strongly depended on the location of the glandular tissue.     

核素动态显像评价正常颌下腺功能

目的研究病理状态下的颌下腺功能,须对正常颌下腺功能进行测定。方法用发射型计算机断层（ｅｍｉｓｉｏｎｃｏｍｐｕｔｅｄｔｏｍｏｇｒａｐｈｙ,ＥＣＴ）动态显像技术对１０侧颌下区本底与颞部本底进行了比较研究,并对１１对正常颌下腺腺体功能进行测定。结果２．１８倍的颞部本底可以近似代替颌下区本底;正常颌下腺的摄取指数和分泌指数分别为（１．４２±０．８９）％和（１０９．４±３９．８）％,摄取指数率和分泌指数率分别为（７１．０±１５．２）％和（７７．２±１７．２）％,功能指数为（８９．９±７．４）％。结论应用颌下区本底作为颌下腺功能评价指标更能真实反映腺体的功能,并具有较高的敏感性,取得正常功能范围值为研究影响颌下腺腺体功能的疾病奠定了基础。     

PCNA在db/db自发性糖尿病小鼠颌下腺的表达

目的:观察db/db自发性糖尿病小鼠颌下腺的病理形态学改变,增殖细胞核抗原(PCNA)在糖尿病小鼠颌下腺中的表达。方法:选取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的db/ m正常小鼠颌下腺,应用HE染色及免疫组织化学方法染色后进行图象分析,统计PCNA在颌下腺组织内表达的细胞阳性率。结果:随着糖尿病的发展,颌下腺组织出现腺体萎缩及及颗粒曲管数目减少,实质细胞排列不整齐,呈簇状堆集,结缔组织、纤维及血管增多。PCNA在对照组及糖尿病组颌下腺中均有表达,对照组PCNA细胞阳性率高于糖尿病组。糖尿病组PCNA细胞阳性率随病程延长呈减弱趋势,且减少明显快于对照组。结论:①db/db糖尿病可导致颌下腺组织萎缩及实质细胞形态学改变。②PCNA表达逐渐减弱,较对照组明显降低,说明糖尿病可能导致颌下腺腺体增殖活动的减弱。     

Carbachol-induced in vitro secretion of certain human submandibular proteins investigated by mass-spectrometry

Objective

To investigate protein content of saliva produced in vitro by samples of human submandibular gland following stimulation with the muscarinic agent carbachol.

Design

Tissue samples, obtained at surgery from seven patients and showing normal morphological appearance, were tested for 30 min: in absence of carbachol and atropine; in presence of carbachol (10 μM); in presence of carbachol (10 μM) and atropine (20 μM); or in presence of just atropine (20 μM). Medium was analysed by high-performance liquid chromatography-mass-spectrometry. Neither before nor during surgery were the patients exposed to drug treatments that were likely to influence the in vitro secretion.

Results

Proline-rich proteins (PRP)-1 and -3, peptide PC and PB, statherin, cystatins SN, S1 and S2 were invariably found in control gland tissue medium. Mean concentrations of these proteins/peptides in the medium were non-proportionally elevated following carbachol exposure to the gland tissues. Difference between basal release and carbachol-induced secretion achieved statistical significance as to all the proteins/peptides under study but for statherin. Atropine alone or atropine plus carbachol caused no significant changes compared to the basal release of proteins/peptides.

Conclusions

In vitro studies on salivary glands make it possible to study protein secretion from individual glands and thus, to reveal the contribution of the various types of gland to protein/peptide content of whole saliva. The disproportional responses to carbachol may imply that the proteins/peptides are not confined to the same cells or to the same intracellular locations and are therefore not secreted as packages at parasympathetic cholinergic activity.     

Carbachol improves secretion in the early phase after rabbit submandibular gland transplantation

Oral Diseases (2010) 16 , 351–359 Objectives: To investigate the changes in the muscarinic receptor signaling pathway with submandibular gland (SMG) transplantation and whether carbachol improves secretion in transplanted SMGs. Materials and methods: SMG autotransplantation was performed in a rabbit model. Carbachol (1 μM) was infused into the transplanted glands from postoperative day 1–7. The expression of the M1 and M3 muscarinic receptors, aquaporin‐5 (AQP5), and phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK1/2) was measured by RT‐PCR, immunoblotting or immunofluorescence. The content of inositol 1, 4, 5‐trisphosphate (IP₃) was measured by radioimmunoassay. Results: Salivary flow of the transplanted SMGs was decreased after transplantation. As well, the expressions of M1 and M3 receptors and their downstream signaling molecules, IP₃, p‐ERK1/2 and AQP5, were all reduced. Atrophy of acinar cells was shown in transplanted glands. However, all these alterations were reversed after carbachol treatment for 7 days. Furthermore, carbachol directly increased the mRNA expression of AQP5 and phosphorylation of ERK1/2 in cultured neonatal rabbit SMG cells. Conclusion: A lack of acetylcholine and downregulation of the muscarinic receptor signaling pathway is involved in the early hypofunction of transplanted SMGs. Carbachol treatment could be a new therapeutic strategy to improve secretion and prevent the obstruction of Wharton’s duct in the early phase after SMG transplantation.