Formation and fate of ethionine-induced cytoplasmic crystalloids in rat parotid acinar cells.

The formation and fate of cytoplasmic crystalloids in rat parotid acinar cells were investigated during ethionine intoxication and recovery. By day 3 of ethionine treatment, acinar cells had numerous autophagic vacuoles containing recognizable secretory granules and fragments of rough endoplasmic reticulum. By day 5, immature crystalloids were present in many of the autophagic vacuoles, and as the crystalloids matured, a 7-nm periodicity became apparent. Crystalloids were never observed in the Golgi saccules or in any other organelle associated with secretory granule formation. When ethionine treatment was stopped, the acinar cells rapidly returned to their normal morphology. The majority of the crystalloids and autophagic vacuoles were lost from the cells during the first two to three days of recovery. At this time annulate lamellae were present intracellularly, and macrophages, many containing crystalloids, were associated with the basal surface of the acinar cells. These results indicate that the cytoplasmic crystalloids are formed in autophagic vacuoles, and do not represent an abnormal secretory product. Additiontionally, during recovery crystalloids may be removed from the acinar cells by interaction with macrophages. The sequence of autophagic vacuole formation, development of crystalloids, macrophage infiltration and phagocytosis of acinar cell debris appears to be a non-specific response of the rat parotid gland to cellular injury occurring in a variety of experimental and pathological conditions.     

Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.     

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.     

Isolation and characterization of a new Ca2+/calmodulin-dependent protein kinase from isoproterenol-stimulated proliferating rat parotid acinar cells.

A new Ca2+/calmodulin-dependent serine kinase was isolated from rat parotid gland acinar cells following chronic treatment with the beta-agonist isoproterenol. A single-step purification was performed on a calmodulin-agarose affinity column, following solubilization with Triton X-100. Among various substrates tested, bovine galactosyltransferase was the preferred substrate of the kinase, followed by glycogen synthetase greater than histone greater than phosphodiesterase greater than phenylalanine hydroxylase greater than phosphorylase b greater than bovine serum albumin. In comparison, a spleen preparation of Ca2+/calmodulin-dependent kinase did not show galactosyltransferase to be the preferred substrate. Thus, the enzyme would appear to be similar to the human galactosyltransferase-associated kinase. The kinase activity was saturable with 100 microM Ca2+ and 2 microM calmodulin. The molecular mass determined by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoreses was 75 kDa with a pI of 4.3. The Vmax was 3500 mumol/(min.mg protein) with a Km of 1.6 microM for the transferase substrate. Leukotriene C and prostaglandin E2 were found to be specific noncompetitive inhibitors of the rat galactosyltransferase-associated kinase.     

Uptake of horseradish peroxidase in spontaneous adenomas of the rat pituitary

The uptake of horseradish peroxidase was investigated by electron microscopy in 9 non-tumorous adenohypophyses and 13 spontaneous pituitary adenomas of adult female Long-Evans rats. Pituitary adenoma cells retained their ability for endocytosis and exhibited more extensive deposition of horseradish peroxidase than non-tumorous adenohypophysial cells.     

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.     

Sphingosine stimulates calcium mobilization in rat parotid acinar cells

In fura-2-loaded parotid acinar cells, 50-200 microM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 microM sphingosine failed to increase [Ca2+]i, but 100 or 200 microM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H-7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca(2+)-influx and further stimulates the release of Ca2+ from agonist-sensitive intracellular pools by a mechanism that is independent of protein kinase C.     

Uptake of horseradish peroxidase by bone cells during endochondral bone development

Summary To investigate the mechanisms whereby bone cells absorb organic bone-matrix components during endochondral bone development, rat humeri were examined, employing horseradish peroxidase as a soluble protein tracer.Intravenously-injected peroxidase filled the osteoid layer and penetrated into the osteocyte lacunae and canaliculi, but did not enter the mineralized bone matrix. Whereas osteocytes rarely took up exogenous peroxidase, osteoblasts and osteoclasts actively endocytosed peroxidase in pinocytotic coated vesicles, tubular structures, and vacuoles. They also formed endocytotic vacuoles containing peroxidase in the Golgi area. The Golgi apparatus and dense bodies of these bone cells were, however, free of reaction products. Osteoclast ruffled borders were responsible for peroxidase absorption. In the osteoblast, osteocyte and osteoclast, endogenous peroxidatic reaction was detected only in mitochondria and not in other membrane-bounded vesicles and bodies. These results strongly suggest that both osteoblasts and osteoclasts participate in the resorption of bone-matrix organic components during bone remodelling.     

Changes in the content and distribution of sialic acid on the basal surface of isoproterenol-stimulated mouse parotid acinar cells

The presence, distribution and content of sialic acid on the cell surface in collagenase-dispersed acini obtained both from unstimulated as well as from in vivo isoproterenol-stimulated mouse parotid have been studied. To this end, sialic acid residues have been qualitatively and quantitatively analyzed by 1) cytochemical labeling by wheat germ agglutinin (WGA), 2) biochemical procedures and 3) isotopic labeling by [3H]WGA (WGA-N-[acetyl-3H]-acetylated). Electron microscopy revealed striking differences in the binding of ferritin-conjugated WGA at the basal, lateral and apical cell surface. Unstimulated acinar cells showed a heavy patch-distributed binding of ferritin-conjugate on the basal cell surface while it was homogeneous and very scarce on the lateral one and absent on the apical cell surface. During the first few hours after isoproterenol, the WGA binding sites at the basal cell surface became homogeneously distributed. This fact was coincident with a loss of about 60 to 70% both in the content of neuraminidase-releasable sialic acid and in the binding of [3H]WGA to the acinar surface. These findings suggest that the release of sialic acid as free residues, which has been involved in the isoproterenol-triggered cell proliferation-inducing mechanism in the mouse parotid, would occur at the glycocalyx corresponding to the basal plasma membrane of the acinar cells.     

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.     

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.     

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.     

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.     

Calcium metabolism and amylase release in rat parotid acinar cells.

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10muM), isoproternol (1 or 10 MUM), phenylephrine (1 muM), carbamoylcholine (0.1 or 1 muM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (greater than 3h) of probably a similar dimension. Cholinergic and alpha-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas beta-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (beta-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).     

Alcohol potentiation of isoproterenol-stimulated cyclic AMP accumulation in rat parotid

The ability of beta-adrenergic agonists to elevate rat parotid cyclic AMP concentrations is potentiated greatly by certain organic solvents. Propanol was found to be more effective than other tested solvents. Propanol stimulated adenylate cyclase and inhibited low Km cyclic AMP phosphodiesterase activities however the magnitude of effect upon these enzyme activities probably does not account for the potentiation of cyclic AMP accumulation observed in intact cells.