N-linked protein glycosylation in the rat parotid gland during aging

N-Linked protein glycosylation was examined in vitro in dispersed rat parotid acinar cells from young adult (3-6 months) and aged (22-24 months) rats. A small decrease in general protein production was observed with cells from aged animals (approximately 20% lower incorporation of [14C]leucine into 10% CCl3 COOH insoluble protein during continuous pulse labeling). Incorporation of [3H]mannose into N-linked glycoproteins by aged cells was further reduced (approximately 35%). Similarly microsomal membranes from parotid glands of aged animals showed approximately 50% reduction in the synthesis of mannosylphosphoryl dolichol, a key intermediate in the dolichol pathway of protein N-glycosylation. Man-P-Dol synthase, the microsomal enzyme responsible for production of this saccharide-lipid, displayed no change in apparent Km for GDP-mannose when preparations from aged animals were utilized, but did show approximately 50% reduction in Vmax. Following beta-adrenoreceptor activation, cells from both young adult and aged glands showed increased N-linked protein glycosylation almost to the same extent (approximately 2-fold). The data suggested that in aged rat parotid cells there is a basal reduction of activity in the pathway responsible for asparagine-linked protein glycosylation, but that following exocytotic stimuli this pathway responds in a manner comparable to cells from young adult glands.     

Changes in the properties of secretory granules in the palatine gland acinar cells of the postnatally developing rat

This study was designed to examine whether or not phospholipid is contained in the secretory granules of the rat palatine gland acinar cells, and if present, to examine the movements of phospholipid in the secretory granules during postnatal development. The palatine glands of male Wistar rats aged 0 to 56 days were used. Acid-hematin staining showed a few positive acinar cells with a faint reaction in the acini on day 0, numerous positive cells with an intense reaction on day 7, a weakening reaction in the cells on day 14, and almost no reactivity on day 35 and after. In contrast, alcian blue staining showed acinar cells with a weak reaction on day 7, a gradual increase in the reaction from day 14, and the presence of many cells with an intense reaction on day 28 and after. Electron probe microanalysis (EPMA) revealed a higher density of phosphorus in samples on day 7 than on day 56. These findings suggest that developing rat palatine gland acinar cells contain phospholipid in the secretory granules, being particularly more conspicuous around postnatal day 7, but that the amount of phospholipid decreases as the cells change to mature mucous cells.     

Beta-adrenergic regulation of rat parotid gland exocrine protein secretion during aging

beta-Adrenergic regulation of exocrine protein secretion from the parotid gland was studied over the adult rat life span. Enzymatically dispersed cell aggregates were prepared from 3-, 6-, 12-, and 24-month-old rats and exocrine protein secretion (amylase release) measured. No age differences were seen in the time course of amylase release following (-)-isoproterenol stimulation or in the (-)-isoproterenol dose-response curve. the beta-adrenergic antagonist (+/-)-propanolol inhibited (-)-isoproterenol-stimulated protein secretion from parotid cell aggregates of young and old animals equally. Similarly dibutyryl cyclic AMP induced comparable rates of protein secretion from cells of different aged rats. Direct examination of beta-adrenergic receptor characteristics in parotid gland membranes from 3- to 24-month-old rats revealed no differences in the equilibrium dissociation constant (Kd) or maximum specific ligand binding capacity (receptor number). These results suggest that the rat parotid beta-adrenergic system remains functionally intact throughout the animals' lifetime.     

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.     

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.     

Characteristics of stimulated parotid gland secretion in the aging rat

The production of pilocarpine-stimulated parotid saliva was evaluated in young adult and aged male and female rats. Parotid salivary flow rate was about 50% lower in aged animals of both sexes. Saliva of aged animals had the same Na+ concentration as that of young rats but contained about 40% more protein. Salivary K+ concentration was similar in young and aged males but not females.     

Investigation of the capillaries of the rat parotid gland during a 3-h secretory cycle

Principles governing changes in diameter of the lumen and area of the endothelium of capillaries in the parotid salivary gland of rats with an experimentally established 3-h secretory cycle, and also during circulatory ischemia of the organ for 5 min, were studied. The diameter of the lumen and area of the endothelium of the capillaries changed very little with the phase of the secretory cycle. Occlusion of the common carotid artery for 5 min likewise did not change the diameter of the lumen or the area of the endothelium of the capillaries. The results suggest that the increase in the transcapillary blood flow during secretion can be explained by the recruiting of more capillaries into the circulation.Laboratory of Electron Microscopy, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 7, pp. 104–106, July, 1975.     

Effects of ammonium chloride on membrane currents of acinar cells dispersed from the rat parotid gland

In acinar cells freshly dispersed from rat parotid glands, the effects of ammonium chloride (NH₄Cl) on membrane currents were studied using the whole-cell clamp method. When membrane currents were recorded with command pulses to 0 mV, applied at 2-s intervals from a holding potential of –70 mV, NH₄Cl (5–20 mM) transiently decreased outward currents and then slowly increased both outward and inward currents. After reaching a peak in about 40–50 s, both outward and inward currents gradually decreased in the presence of NH₄Cl and, on its wash-out, the currents returned to the control level. Butyrate (5–20 mM) had little effect on the resting membrane currents, but markedly inhibited the response to NH₄Cl. Tetraethylammonium (5 mM) strongly reduced both the resting and NH₄Cl-induced outward currents, whereas it slightly potentiated the NH₄Cl-induced inward current without affecting the membrane current at the holding potential. Without ATP in the patch pipettes, carbachol-induced membrane currents were relatively resistant to Ca²⁺ removal from the external medium, but NH₄Cl-induced currents were quickly abolished in the absence of Ca²⁺. We conclude that intracellular alkalinization with NH₄Cl increases Ca²⁺ influx and activates Ca²⁺-dependent outward K⁺ and inward Cl^– currents.     

The secretory granules of the acinar cells of the mouse submaxillary gland

Absence of membranes from the secretion granules of the acinar cells of the submaxillary gland of the mouse had led to speculation concerning mechanisms of secretion of these cells. By means of rapid perfusion fixation, smooth membranes have been identified around the secretion granules, and the mode of secretion proves to be similar to that of the other exocrine glands. The evidence suggests that potent membranolytic agents of unknown nature, capable of rapidly destroying the membranes are present in these secretory granules.     

Changes in pancreatic acinar cell nuclear number and DNA content during aging in the rat

Pancreatic acinar cells from rats 5 to 658 days (94 weeks) of age were isolated by enzymatic dissociation and stained with the DNA specific fluorochrome Hoechst 33258. The nuclear DNA content and the incidence of binucleation were estimated in these cells. Total pancreatic weight, RNA, protein and DNA, and the incorporation of ³H-thymidine into pancreatic acinar cell DNA were also estimated in similar animals as measures of pancreatic growth. From 5 to 17 days after birth, 95% of the cells were mononucleate diploid and 5% were binucleate diploid; but during the period of rapid pancreatic growth over the following 39 days, acinar cells became increasingly binucleate. By 56 days after birth, 64% of cells were binucleate with a diploid DNA content per nucleus; and the incidence of binucleation then remained constant. At 28 days of age, 4% of mononucleate cells were tetraploid, increasing to 6% at 658 days of age. At this time 3% of binucleate cells contained dual tetraploid nuclei. There is thus a rapid development towards diploid binucleate acinar cells in the growing, postnatal pancreas; and in the adult pancreas a small proportion of these cells develop tetraploid nuclei.     

Proliferation and phenotypic preservation of rat parotid acinar cells

The purpose of this study is to develop an initial step in salivary gland tissue engineering through proliferation and phenotypic preservation of rat parotid acinar cells in vitro. By using the explant outgrowth technique and M199 medium with the addition of sialic acid, acinar cells not only survived for more than 30 days in the absence of basement membrane substrates but also proliferated to yield cells with acinar phenotypic expression. Furthermore, we tested whether chitosan can be used as a synthetic extracellular matrix to culture salivary acinar cells. Chitosan is a deacetylated product of chitin, which is a plentiful polysaccharide found in nature and is safe for the human body, but little is known about the utility of chitosan in culturing salivary acinar cells. It was found that coating fibronectin on chitosan membrane improved the attachment of acinar cells in the initial stage. However, the poor attachment of acinar cells on pure chitosan membrane did not affect cell growth after longer culture times, indicating that chitosan is potentially useful as a tissue-engineering scaffold of the salivary gland. These in vitro results are encouraging because such a culture system may serve as an artificial salivary gland for future use in the treatment of patients with salivary hypofunction.     

Non-specific secretory supersensitivity in rat parotid gland following neonatal sympathetic denervation

Newborn rats were surgically sympathectomized by extirpation of the left superior cervical ganglion. After 9 weeks the parotid glands of both sides were used for secretory studies. Isoprenaline, dopamine, and the dibutyryl analogue of cAMP (DBcAMP) caused an increase in amylase release, which was significantly higher in the denervated glands. Also carbamylcholine was more effective in the denervated gland; the concentration-response curve was shifted to the left, and the maximal output of amylase was increased. Neonatal sympathetic denervation induces supersensitivity for both adrenergic and cholinergic agonists as well as for DBcAMP. This may be due to compensatory mechanisms involving both up-regulation of receptors as well as amplification of the intracellular mediation.     

Differentiation of myoepithelial cells in the developing rat parotid gland

Parotid glands of rats were prepared for light and electron microscopy and for the histochemical demonstration of myofibrils and alkaline phosphatase (AkPase) activity. Through 18 days in utero, the epithelial cells of the developing gland remain relatively undifferentiated. At 20 days in utero, a few cells in the outer layer of the terminal buds and adjacent segments of ducts acquire a cilium, the initial indication that they are differentiating into myoepithelial cells (MEC). Up until the time of birth, the only additional characteristics of MEC that the outer cells develop are to flatten against the underlying cells, begin to send out processes, and produce a few dilated cisternae of rough endoplasmic reticulum. Myofibrils and AkPase activity are first detected at the light microscopic level at five days after birth, around both the developing acini and intercalated ducts. Progressive increases in AkPase activity and in the size and number of myofibrils continue until the acini and intercalated ducts are invested with well-differentiated MEC at 15 days. Subsequently, as the acini undergo maturation during the weaning period (18-25 days), the MEC cease to surround the acini and assume the adult pattern of investing only the intercalated ducts. The pattern of MEC differentiation in the parotid gland differs from those in the sublingual and submandibular glands of the rat in several important respects. They begin to differentiate last, yet mature almost as early as do the MEC of the sublingual gland; they begin to differentiate prior to, rather than simultaneously with, the secretory cells; and their distribution changes as the acinar cells become mature.     

Developmental changes of sugar residues and secretory protein in mucous cells of the early postnatal rat parotid gland.

Mucous cells have been identified in the terminal portions of the early postnatal parotid gland in human and rat, although mature parotid gland acini are composed of serous cells or seromucous cells. Previously, Ikeda et al. demonstrated that mucous cells are present in the rat parotid gland on days 1 to 8 after birth and that the secretory granules within these mucous cells share some histochemical characteristics with mature serous cells. However, it is still not clear whether the mucous cells change into serous cells as the gland develops. The purpose of this study was to determine whether the mucous cells that appear in the early postnatal rat parotid gland change into serous cells. Parotid glands were obtained from male or female Wistar rats (aged 0-14 days and adults). Fixed tissue sections were reacted with soybean agglutinin (SBA) and wheat germ agglutinin (WGA) to detect glycoconjugates, or were stained using an anti-neonatal submandibular gland protein B1 (SMG-B1) antibody to identify serous acinar cells. The sections were observed by transmission electron microscopy. Electron microscopy revealed that cells with characteristics intermediate between those of mucous and serous cells (transitional cells) appeared around day 8 and that the nuclei of these cells did not show chromatin condensation, a characteristic of apoptotic cells. Lectin histochemistry showed that the mucous cells had the same sugar residues as the serous cells, which appeared after day 10. Immunohistochemistry with an anti-SMG-B1 antibody gave a positive reaction not only in the cells with highly electron-dense granules but also in the electron-dense cores of bipartite or tripartite granules in the transitional cells. Cells with morphological characteristics intermediate between those of mucous and serous cells (transitional cells) appearing in the early postnatal rat parotid gland begin to produce B1-immunoreactive protein common to serous acinar cells during development of the gland.     

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 contaning 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. Additionally, 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.     

Membrane potential measurement in parotid acinar cells

1. Intracellular recording of membrane potential was made from acinar cells of the isolated mouse parotid gland superfused with physiological salt solutions.2. The mean acinar resting membrane potential was - 68.5 mV during superfusion with Krebs-Henseleit solution. Shift of the superfusion solution to one containing ACh or adrenaline (10(-5)M) always caused a transient hyperpolarization (about 10-15 mV).3. The membrane potential was mainly dependent on the extracellular K concentration ([K](o)). Increasing [K](o) tenfold decreased the membrane potential by 50 mV. This depolarization was not mediated by ACh release from depolarized nerve endings, since it was seen in the presence of atropine (1.4 x 10(-6)M) and not caused by the accompanying reduction in [Na](o) to 40 mM caused only a small depolarization (less than 10 mV).4. When the superfusion solution was shifted, during intracellular recording, from a normal Krebs-Henseleit solution ([K] = 4.7 mM) to a K-free solution, a hyperpolarization of about 8 mV was measured. Reintroduction of the normal K-containing solution after a longer period of K deprivation (30-70 min) resulted in a short-lasting pronounced hyperpolarization (about 20 mV) which could be blocked by Strophanthin-G (10(-3)M).5. In contrast to previous reports, the present findings indicate that the membrane potential of salivary acinar cells is similar, with respect to magnitude and K-dependence, to that of cells of more thoroughly investigated tissues, such as muscle and nerve, and that the membrane Na-K pump is electrogenic, at least when the cells have been loaded with Na.