The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].     

G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction

The mechanism by which G protein-coupled receptors (GPCRs) translate extracellular signals into cellular changes initially was envisioned as a simple linear model: activation of the receptor by agonist binding leads to dissociation of the heterotrimeric GTP-binding G protein into its alpha and betagamma subunits, both of which can activate or inhibit various downstream effector molecules. The plethora of recently described multidomain scaffolding proteins and accessory/chaperone molecules that interact with GPCR, including GPCR themselves as homo- or heterodimers, provides for diverse molecular mechanisms for ligand recognition, signalling specificity, and receptor trafficking. This review will summarize the recently described GPCR-interacting proteins and their individual functional roles, as understood. Implicit in the search for the functional relevance of these interactions is the expectation that enhancement or disruption of target cell-specific events could serve as highly selective therapeutic opportunities.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.     

Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland.

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.     

Beta adrenergic and muscarinic receptor densities of rat submandibular main duct

[3H]DHA binding studies show that main duct of rat submandibular gland has both beta 1 and beta 2 adrenoceptors, with the percentages of each being 69 and 31%, respectively, whereas whole submandibular gland has 90% beta 1 and 10% beta 2 adrenoceptors. Muscarinic receptors of main duct are 25% less than that of whole submandibular gland.     

Taurine in submandibular gland of the rat: effect of muscarinic drugs.

Taurine exerts a number of actions in mammalian cells, including regulation of ion transport and osmoregulation. The production and secretion of saliva involve transepithelial ion transport, thereby making the plasma-like primary saliva hypotonic before secretion. Therefore, it is plausible to suggest modulation of salivary taurine by muscarinic agents that affect salivary gland function. One of the objectives of this study was to determine tissue content and localization of taurine in the submandibular gland of the rat. Further, we determined whether treatment with muscarinic drugs that either increase (e.g., pilocarpine) or decrease (e.g., propantheline) saliva secretion affects the submandibular gland taurine content. The results indicate that the submandibular gland contains an appreciable amount of taurine (8.9 +/- 0.3 micromoles/g wet wt). Further, acute treatment of the rats with either of the muscarinic drugs did not significantly affect tissue taurine content compared to the control group. By contrast, chronic treatment with propantheline, but not pilocarpine, reduced the tissue content of taurine compared to the control rats (p<0.05). Utilizing light microscopic immunohistochemical techniques, intense immunoreactivity was found primarily in the striated ducts of the submandibular gland. Neither pilocarpine nor propantheline treatment led to differential distribution of immunoreactivity in this tissue. In conclusion, the submandibular gland contains an appreciable amount of taurine, primarily in the striated ducts, that can be decreased by chronic muscarinic receptor blockade.     

Function of a heterologous muscarinic receptor in T cell antigen receptor signal transduction mutants

Previously we have described a system of somatic cell genetics (J.CaM1 and J.CaM2) for analyzing signal transduction via the T cell antigen receptor complex (CD3/Ti). Here we describe a third mutant, J.CaM3, which also expresses high levels of receptors that are functionally impaired. Like J.CaM1, J.CaM3 demonstrates partial signal transduction via CD3/Ti to only certain stimuli. J.CaM1, J.CaM2, and J.CaM3 define three non-Ti complementation groups involved in receptor function. To evaluate the mutations further we have introduced a heterologous receptor, the human muscarinic receptor 1 (HM1), into the parental Jurkat and mutant cell lines. This receptor demonstrates signal transduction competence in all these hosts, indicating that 1) T cells express the necessary apparatus for the coupling of HM1 to second messenger generation and 2) the mutations in the J.CaM family all affect molecules that are specific to CD3/Ti, and not HM1, function. Finally, the HM1 receptor exhibits partial sensitivity to cholera toxin in Jurkat cells, in contrast to the virtually complete sensitivity of CD3/Ti to cholera toxin.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction

M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Homer蛋白介导谷氨酸受体信号转导

Homer蛋白是一类联系突触内细胞骨架蛋白、信号蛋白的重要物质。Homer家族蛋白可和mGluRI、IP3R、Shank、RyR中富含脯氨酸的序列结合。Homer蛋白可以自我交联形成同聚或异聚体 ,此多聚体通过与多种蛋白、受体形成复合体并相互作用 ,在信号转导、突触形成、受体在细胞定位起重要作用。     

Characterization of nontransformed and transformed androgen receptor from rat submandibular gland

Rat submandibular gland cytosol contained androgen receptor which had a single class of specific binding and an apparent dissociation constant of (1.1-1.2) X 10(-9) M. The process of transformation was investigated by a slightly modified minicolumn method in which the transformed receptor complexes were separated from the nontransformed receptor and meroreceptor. 10 mM ATP or pyrophosphate at 0 degrees C induced transformation of androgen receptor as did heat or salt treatment. 20 mM of sodium molybdate completely inhibited transformation that resulted from ATP, heat or salt treatment. The nontransformed androgen receptor complexes sedimented at 8 S and eluted at 250-260 mM KCl from DEAE-Sephacel, and its molecular weight was found to be 220 000 on Sephacryl S300 gel chromatography. On the other hand, the transformed androgen receptor complexes sedimented at 4.1-4.3 S (ATP or KCl treatment) or 3.5-3.8 S (heat treatment) and eluted at 60-80 mM KCl from DEAE-Sephacel. The molecular weight of the transformed androgen receptor complexes was 80 000-85 000 (ATP or KCl treatment) or 70 000-80 000 (heat treatment). These results suggest that the transformation of androgen-receptor complexes from rat submandibular gland was induced by the subunit dissociation and that salt bridges may be involved in the subunit interaction.     

Mode of stimulatory action of deoxycholate in signal transduction system of isolated rat pancreatic acini.

Mode of stimulatory action of deoxycholate (DCA) on the secretagogue-induced amylase release and the phospholipase C reaction in isolated rat pancreatic acini was investigated using sodium fluoride (NaF), which is a direct activator of GTP-binding proteins (G proteins). DCA enhanced the amylase release induced by submaximal concentrations of NaF without affecting the maximal level of this reaction. Under the similar conditions, DCA enhanced the NaF-induced phospholipase C reaction. These stimulatory effects of DCA on the NaF-induced amylase release and phospholipase C reaction are comparable to those on the secretagogue-induced reactions reported previously. These results suggest that DCA acts on the coupling of a G protein(s) to the phospholipase C in the membrane transduction mechanism in isolated rat pancreatic acini.     

Isoproterenol-induced desensitization of mucin release in isolated rat submandibular acini

Release of [14C]glucosamine-labelled mucins was studied in vitro using well-characterised preparations of rat submandibular acini. Mucin release was stimulated by forskolin, an activator of the catalytic subunit of adenylate cyclase, and 3-isobutyl-1-methylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor. Both stimulated in a dose-dependent manner to the same maximum as that seen with isoproterenol. Neither forskolin nor IBMX added in the presence of isoproterenol increased secretion above the maximum in response to isoproterenol alone, suggesting a similar mechanism of action, mediated by cyclic AMP. Prior exposure of acini to isoproterenol (10 microM) for 45 min, followed by washout resulted in (a) persistent increase in basal secretion which was abolished by propranolol and (b) reduced stimulation of mucin secretion in response to either a second isoproterenol challenge, noradrenaline or forskolin. Thus, exposure of rat submandibular acini in vitro desensitizes the cells to subsequent stimulation. Although this mimics the decreased beta-adrenergic secretory responses seen in submandibular cells from cystic fibrosis patients, results suggest that the isoproterenol-induced desensitization is at the level of beta-receptor and adenylate cyclase, rather than distal to cyclic AMP.     

Ribonucleic acid association with androgen receptor from rat submandibular gland

The transformed androgen receptor from rat submandibular gland converts to a faster sedimenting form (6-8S) on a glycerol gradient centrifugation after withdrawal of a transformation-inducing reagent (KCl or ATP). In this report, the association of cytosolic RNA with the transformed androgen receptor was investigated as a possible mechanism of molecular conversion of the androgen receptor. When the transformed and converted androgen receptors were treated with RNase A, these receptors sedimented at 4.5S in a low-salt glycerol gradient. Addition of RNA from rat submandibular gland to the RNase-Sepharose-treated transformed receptor caused a shift of receptor peak from 4.5S to 5.8S. RNA from rat submandibular gland, yeast RNA and E. coli rRNA inhibited DNA-cellulose binding of a RNase-treated transformed receptor in the absence of molybdate. These observations suggest that conversion from the transformed 4S androgen receptor to a 6-8S form resulted from the association of RNA(s) with the transformed receptor.     

Immunolocalization of myoepithelial cells in isolated acini of rat exorbital lacrimal gland: Cellular distribution of muscarinic receptors

The secretion of proteins and fluids from the exorbital lacrymal gland of rat is mainly controlled by muscarinic receptors. In a recent pharmacological study, Mauduit et al (Am J Physiol (1993) 264, C1550–C1560) identified a homogeneous population of M₃ muscarinic receptors in preparations of acini from these tissues. In order to define the cellular composition of these acini and localize the muscarinic receptors; we have performed an immunofluorescent labelling study combined with confocal scanning microscopy. Antibodies raised against components of the different cytoskeletal networks (α-smooth muscle actin, cytokeratin peptide 14 and α-tubulin) revealed the presence of two different cell types. Cells with a stellate form are identified as myoepithelial cells, whereas rounded cells are secretory acinar cells. Both cell types are reactive with an antibody specifically directed against the muscarinic receptor. However, myoepithelial cells appear more intensely labelled than acinar cells. The roles of myoepithelial cells and secretory cells in the physiological function of the gland are discussed in terms of the distribution of muscarinic receptors.