Vitamin D receptors in isolated rat parotid gland acinar cells

Rat parotid gland was examined for the presence of 1α,25-dihydroxycholecalciferol receptors using sucrose density gradient ultracentrifugation techniques. [³H]DHCC bound specifically and with high affinity to a 3.2 S protein present in nuclear and cytosolic fractions of isolated parotid acinar cells. Values for the equilibrium dissociation constant and for the receptor concentration were determined to be approx. 0.1 nM, and 12 fmol/mg protein, respectively. In competitive inhibition experiments, the 3.2 S protein displayed 100-fold lower affinity for 25-hydroxycholecalciferol than for DHCC, and did not bind estradiol or methylprednisolone. These results suggest that rat parotid gland acinar cells contain classical DHCC receptors. A similar approach failed to provide evidence of DHCC receptors in isolated pancreas acinar cells, lacrimal gland or submandibular gland. It has been previously reported that vitamin D is essential for normal exocrine secretion from the rat parotid gland (Tenenhouse, A. and Afari, G. (1978) Biochim. Biophys. Acta 538, 631–634). The present findings suggest that this effect is the result of a direct action of DHCC on the parotid gland acinar cell. The absence of DHCC receptors in other exocrine cells suggests that tissue sensitivity to DHCC is not a general property of exocrine systems.     

The relationship between the incorporation of 32P into phosphatidic acid and phosphatidylinositol in rat parotid acinar cells

Muscarinic and α-adrenergic stimulation of rat parotid acinar cells increases the turnover of phosphatidylinositol and phosphatidic acid. It is thought that this is initiated by hydrolysis of phosphatidylinositol, which would predict an increase in ³²P incorporation into phosphatidic acid before phosphatidylinositol. We have demonstrated an increase in ³²P incorporation into the former within 1 minute and into the latter by 2 minutes. The initial rapid rate of ³²P incorporation into phosphatidic acid slows, and the ³²P content reaches a steady state after 15 minutes. During the first 2 minutes after the addition of atropine to carbamylcholine stimulated cells, ³²P is lost from phosphatidic acid, and an equal amount is gained by phosphatidylinositol, after which ³²P incorporation equals that of the control. In cells prelabelled with ³²P, carbamylcholine, in the presence of oligomycin stimulated the loss of ³²P from phosphatidylinositol but had no effect on phosphatidic acid.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

Transferrin secretory pathways in rat parotid acinar cells

Transferrin is the major iron transporter in blood plasma, and is also found, at lower concentrations, in saliva. We studied the synthesis and secretion of transferrin in rat parotid acinar cells in order to elucidate its secretory pathways. Two sources were identified for transferrin in parotid acinar cells: synthesis by the cells (endogenous), and absorption from blood plasma (exogenous). Transferrin from both sources is secreted from the apical side of parotid acinar cells. Endogenous transferrin is transported to secretory granules. It is secreted from mature secretory granules upon stimulation with a β-adrenergic reagent and from smaller vesicles in the absence of stimulation. Exogenous transferrin is internalized from the basolateral side of parotid acinar cells, transported to the apical side by transcytosis, and secreted from the apical side. Secretory processes for exogenous transferrin include transport systems involving microfilaments and microtubules.     

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of beta-adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.     

Effect of proteolytic cleavage on functional properties of muscarinic acetylcholine receptors in rat pancreatic and parotid acinar cells.

Muscarinic acetylcholine receptors in isolated rat pancreatic acinar cells have an apparent Mr of 88 000, which could be decreased to 46 000 by papain, as deduced by covalent binding of the specific alkylating agent [3H]propylbenzilylcholine mustard. Muscarinic receptors on papain-treated acinar cells retained the antagonist-binding site and both high- and low-affinity binding sites for the cholinergic agonist carbachol. Similar results were observed in studies with rat parotid acinar cells, although the receptors in both control and papain-treated cells were each 10 000-15 000 Da smaller than in pancreas. Additionally, muscarinic receptors in papain-treated pancreatic acinar cells retained the ability to mediate carbachol stimulation of digestive-enzyme secretion. These results demonstrate that the characteristic binding properties of muscarinic receptors for both agonists and antagonists as well as their ability to translate agonist occupancy into a physiological response are not altered by proteolytic cleavage.     

Cellular characteristics of long-term cultured rat parotid acinar cells

Summary We have successfully maintained and biochemically characterized differentiated rat parotid acinar cells cultured for long periods (6 mo.). The cells were cultured on a reconstituted basement membrane matrix in a medium containing a variety of agents that promote cellular proliferation and differentiation. The cultured cells retain the characteristics of the parental parotid acinar cells. They exhibit both secretory granules and abundant cellular organelles required for protein synthesis and secretion. In situ hybridization and immunocytochemistry demonstrate high levels of proline-rich protein mRNA and protein, and lower levels of amylase mRNA and protein, in their cytoplasm. These findings suggest that rat parotid acinar cells can be maintained in a differentiated state in vitro for long periods, and can serve as a useful model system for studying the regulation of exocrine secretory processes.     

Characterization of cysteine string protein in rat parotid acinar cells

Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP1_1–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP1_1–82, containing the J-domain only, nor GST-CSP1_83–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.     

Regulation of calcium handling by rat parotid acinar cells

Summary Salivary gland fluid secretion following neurotransmitter stimulation is Ca²⁺-dependent. We have studied the control of cellular Ca²⁺ following secretory stimuli in rat parotid gland acinar cells. After muscarinic-cholinergic receptor activation, cytosolic Ca²⁺ is elevated 4–5 fold, due to both intracellular Ca²⁺ pool mobilization and extracellular Ca²⁺ entry. Fluid movement ensues due to the Ca²⁺-activated enhancement of membrane permeability to K⁺ and Cl^–. Basal cytosolic Ca²⁺ levels are tightly controlled at 150–200 nM through the action of high affinity and high capacity ATP-dependent Ca²⁺ transporters in the basolateral and endoplasmic reticulum membranes. Activity of these Ca²⁺ transporters can be modulated to facilitate rapid responsiveness and a sustained fluid secretory response necessary for alimentary function.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Homologous desensitization of substance-P-induced inositol polyphosphate formation in rat parotid acinar cells.

Maximal concentrations of substance P and methacholine induced a rapid increase in [3H]inositol trisphosphate ([3H]IP3) formation. After about 1 min, the [3H]IP3 in the substance-P-treated cells ceased to increase further, whereas in the methacholine-treated cells [3H]IP3 continued to increase. Addition of methacholine to the substance-P-treated cells caused a rapid increase in [3H]IP3, whereas a second addition of a 10-fold excess of substance P had no effect. Pretreatment of cells with substance P, followed by removal of the substance P by washing, resulted in a decreased response to a second application of substance P. A similar protocol involving pretreatment with methacholine had no effect on subsequent responsiveness to substance P. Analysis of [3H]substance P binding to substance-P-treated cells indicated that the number of receptors for substance P was decreased, but the affinity of the receptors for substance P was unaffected. After substance P pretreatment, a prolonged incubation (2 h) restored responsiveness of the cells to substance P, measured as [3H]IP3 formation, and restored the number of binding sites to control values. These findings indicate that, in the rat parotid gland, substance P induces a homologous desensitization of its receptor, which involves a slowly reversible down-regulation or sequestration of substance-P-binding sites.     

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with ³⁵S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl β-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.     

beta-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by beta-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after beta-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.     

The relationship between synthesis of secretory products and reducing capacity in pancreas and parotid acinar cells

The secretory products in exocrine pancreas acinar cells in utero were found to reduce osmium tetroxide. This reducing capacity was also exhibited by adult pancreas and parotid glands in different phases of synchronized secretion, and after single or chronic administration of a secretagogue, pilocarpine or isoprenaline. In utero, the reducing capacity appeared in the pancreas concomitantly with the synthesis of secretory products, and was limited to the transitional vesicles on the cis Golgi side. After birth, osmium staining occurred in the cis Golgi vesicles and cisternae of both glands. In the chronically-treated parotid gland, where the occupational programme for secretory proteins had been altered, the reducing capacity was diminished, resembling that in embryonic exocrine pancreas.     

Lectin-binding pattern in parotid acinar cells

Summary The localization of complex carbohydrates in the Golgi apparatus, secretory granules and plasmalemma of mouse parotid acinar cells was studied using the fracture-labelling method. The hexose residues of glycoconjugates were identified using ferritin conjugated with Wheat Germ Agglutinin (WGA-), Ricinnus Communis Agglutinin II (RCA-II-), Phaseolus Vulgaris Agglutinin (PHA-) and Limulus Polyphemus Agglutinin (LPA-). We found that the tracture-labelling method allows not only the labelling of membrane faces but also analysis of the compartment's content that is exposed during the fracturing of the tissue. Our results revealed differences in the hexose residues located in the Golgi apparatus, secretory granules and the apical and lateral plasmalemma. Numerous binding sites for WGA-, PHA-and RCA-II-ferritin were demonstrable in the Golgi apparatus. In secretory granules, the WGA-and RCA-II-ferritin binding sites were most numerous, while LPA-ferritin binding sites were very rate. The density of the binding sites for PHA-ferritin showed considerable variation in secretory granules. The apical plasmalemma exhibited a high density of binding sites for all of the lectins used. In the lateral plasmalemma, LPA-ferritin was not bound, and there were fewer binding sites for WGA-, RCA-H-and PHA-ferritin.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells

The effect of extracellular ATP on intracellular free Ca2+ was characterized in quin2-loaded parotid acinar cells. ATP specifically increased the intracellular Ca2+ concentration six-fold above a basal level of 180 nM. Of other purine nucleotides tested, only adenylylthiodiphosphate (ATP gamma S) had significant activity. ATP and the muscarinic agonist carbachol increased intracellular Ca2+ even in the absence of extracellular Ca2+. Both agonists stimulated K+ release, which was followed by reuptake of K+, even in the continued presence of agonist. In the absence of Mg2+, ATP was much more potent but no more efficacious in elevating intracellular Ca2+, suggesting that ATP4- is the active species. The effect of ATP was reversed by removal with hexokinase, arguing against a role for an active contaminant of ATP and against a non-specific permeabilizing effect of extracellular ATP. Lactate dehydrogenase release was unaffected by a maximally effective concentration of ATP. These observations are consistent with a possible neurotransmitter role for ATP in the rat parotid gland.