Delayed inhibition of gap-junctional intercellular communication in the acinar cells of rat submandibular glands induced by parasympathectomy and cholinergic agonists

In this study, the effects of parasympathectomy and cholinergic agonists on gap-junctional intercellular communication and salivary secretion were investigated to clarify the involvement of salivary secretion in delayed uncoupling between acinar cells of rat submandibular glands. Gap-junctional intercellular communication was monitored as dye-coupling in the acinar cells of isolated acini by the transfer of Lucifer Yellow CH. Parasympathectomy induced dye-uncoupling in the acinar cells isolated from denervated salivary glands 12 hr after parasympathectomy-induced salivary secretion. Intraperitoneal application of carbachol (CCh), acetylcholine, pilocarpine, but not isoproterenol, stimulated salivary secretion, and then induced dye-uncoupling in the acinar cells 12 hr later. Atropine suppressed both the salivary secretion and delayed dye-uncoupling induced by parasympathectomy and CCh, when atropine was applied intraperitoneally before the induction of salivary secretion. However, atropine did not suppress the delayed dye-uncoupling by intraperitoneal application of CCh, when atropine was injected after the cessation of CCh-induced secretion. These results suggest that delayed inhibition of gap-junctional intercellular communication by parasympathectomy and cholinergic agonists in rat submandibular glands might be related to the change of secretory function after salivary secretion.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

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.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.     

PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands

We tested the effectof H-89, a protein kinase A (PKA) inhibitor, on the intracellulartransit of the regulated secretory proteins in rat lacrimal glands. Weshow that H-89, by itself, induces the secretion of newly synthesizedproteins trafficking in its presence but not of proteins already storedin the mature secretory granules. This secretion does not depend on thepresence of extracellular Ca²⁺. The proteins released areidentical to those secreted after cholinergic stimulation or under theaction of the ionophore A-23187, but the secretion level is ~40%lower. The effect of H-89 seems to be due to PKA inhibition becauseother protein kinase inhibitors (calphostin C, chelerythrine, H-85) donot induce secretion. We further show that H-89 does not modify therate of glycoprotein galactosylation but induces the secretion of newlygalactosylated glycoproteins. Finally, we used a "20°C block"procedure to show that H-89 affects a trans-Golgi network (TGN)or post-TGN step of the secretory pathway. Our results demonstratethat, in lacrimal cells, H-89 affects the intracellular trafficking ofsecretory proteins, suggesting a role for PKA in this process.

     

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.     

The effects of vinblastine on acinar cells of the exorbital lacrimal gland of the rat

Summary The effects of vinblastine treatment on acinar cells of the rat exorbital lacrimal gland were studied by electron microscopy. Experimental animals of both sexes were given single intraperitoneal injections of (1) vinblastine (4mg/kg body weight) at 1 to 24 h before sacrifice; (2) pilocarpine (20 mg/kg b.w.) for 1 h; or (3) vinblastine for l h followed by pilocarpine for 1 h.Vinblastine treatment caused a number of changes including autophagocytosis, formation of intracisternal granules, and alteration of secretory granules. These changes varied in extent and onset between male and female rats. In addition, the Golgi apparatus was reduced in size and dispersed throughout the cytoplasm. Mitotic figures were commonly observed. Moreover, vinblastine inhibited the pilocarpine-stimulated degranulation of the acinar cells.In view of the known anti-microtubular action of vinblastine, these results suggest that microtubules are involved in various aspects of the transport, packaging, and secretion of exportable proteins in the lacrimal gland. Additionally, autophagocytosis and alteration of secretory granules may partially result from the interaction of vinblastine with membranes.The authors thank Mr. Steve Coriell and Mr. Steve Floyd for preparing the micrographs. Robert Kelly also thanks Dr. George Chapman for his support during the initial phase of this project.     

Effects of cholinergic and α-adrenergic agonists on the monovalent ion content of rat submandibular gland acinar cells studied by X-ray microanalysis

 The effects of cholinergic and α-adrenergic stimulation (in vivo and in vitro) on the monovalent ion content of rat submandibular gland acinar cells were evaluated at the subcellular level by X-ray microanalysis. Fragments of glands or enzymatically dispersed acini were slam-frozen and cut into ultrathin cryosections. Spectra were collected from secretory granules, nucleus, the basal cytoplasm containing endoplasmic reticulum and the apical cytoplasm identified between secretory granules. No significant changes in Na and Cl content were observed after the isolation of acini, but the K concentration decreased compared with cells from in situ glands. The Cl and K content in all four compartments studied decreased significantly after cholinergic stimulation both in vivo and in vitro but in a more restricted fashion after α-adrenergic stimulation. Our findings indicate that: (1) the physiological mechanisms regulating the monovalent ion composition of submandibular cells are relatively well preserved in isolated acinar cells; (2) the results from in vivo experiments are in good agreement with those from in vitro experiments; and (3) the effects of cholinergic and α-adrenergic stimulation on the K⁺ and Cl^–efflux at the subcellular level are similar but the response is generally less with α-adrenergic stimulation. Accepted: 24 April 1997     

Effects of cholinergic and adrenergic agonists on the secretion of fluid and protein by submandibular glands of the guinea-pig and the mouse

Significant differences were observed between the guinea-pig and the mouse in terms of the secretion of fluid, protein and secretory granules from submandibular glands in response to pilocarpine, phenylephrine and isoproterenol. In both the guinea-pig and the mouse, the secretory responses induced by pilocarpine, phenylephrine and isoproterenol were inhibited by pretreatment with 4-DAMP, phentolamine and propranolol, respectively. The results suggest that the submandibular glands of the guinea-pig and the mouse have M₃-cholinoreceptors, as well as α- and β-adrenoceptors, and that these receptors play different roles in the secretion of fluid, protein and secretory granules from guinea-pig and mouse submandibular glands.     

Development of secretagogue response in rat pancreatic acinar cells

Two to 3 days prior to birth, acinar cells of the rat pancreas acquire morphologic and biochemical characteristics of the adult gland. To determine if differentiation of the secretory apparatus coincides temporally with the capacity of the cell to respond to secretory stimuli, lobules of embryonic, neonatal, and adult rat pancreas were compared for their ability to respond to secretagogues presumed to act directly via hormone receptors [caerulein and carbamylcholine (carbachol)] or indirectly (cyclic nucleotide analogs and the Ca²⁺ ionophore A23187). Of all agents tested, only dibutyryl cAMP elicited discharge of secretory proteins at day 20 in utero and preceded hormone stimulation by 1 day. A23187 elicited discharge by Day 21 in utero; its action was near adult levels in contrast to hormonal stimuli whose effect was maximal only at birth. All secretagogues required Ca²⁺ and energy to induce discharge. Pulse-chase autoradiography of lobules from Day 20 embryonic glands indicated that the acinar cells were capable of transporting [³H]leucine-labeled proteins to zymogen granules at rates roughly equivalent to those in adult glands. SDS gel electrophoretograms confirmed that the bulk of ¹⁴C-amino acid incorporation into proteins at a given age was primarily into exportable proteins. The results indicate that acinar cells synthesize and package secretory proteins into zymogen granules about 2 days before they are capable of responding to hormonal stimuli and to intracellular effectors.     

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.     

Fine structure of the silk gland in the mite Ornithocheyletia sp. (Prostigmata,Cheyletidae)

Silk spinning is widely-spread in trombidiform mites, yet scarse information is available on the morphology of their silk glands. Thus this study describes the fine structure of the prosomal silk glands in a small parasitic mite, Ornithocheyletia sp. (Cheyletidae). These are paired acinous glands incorporated into the podocephalic system, as typical of the order. Combined secretion of the coxal and silk glands is released at the tip of the gnathosoma. Data obtained show Ornithocheyletia silk gland belonging to the class 3 arthropod exocrine gland. Each gland is composed of seven pyramidal secretory cells and one ring-folded intercalary cell, rich in microtubules. The fine structure of the secretory cells points to intensive protein synthesis resulted in the presence of abundant uniform secretory granules. Fibrous content of the granules is always subdivided into several zones of two electron densities. The granules periodically discharge into the acinar cavity by means of exocytosis. The intercalary cell extends from the base of the excretory duct and contributes the wall of the acinar cavity encircling the apical margins of the secretory cells. The distal apical surface of the intercalary cell is covered with a thin cuticle resembling that of the corresponding cells in some acarine and myriapod glands. Axon endings form regular synaptic structures on the body of the intercalary cell implying nerve regulation of the gland activity.     

Immunolocalization of AQP-5 in rat parotid and submandibular salivary glands after stimulation or inhibition of secretion in vivo

In vitro studies of cultured salivary gland cells and gland slices have indicated that there may be regulated translocation of aquaporin (AQP)-5 between the apical plasma membrane and intracellular compartments of the secretory cells. However, it remains unknown whether AQP-5 in salivary glands is subject to regulated trafficking in vivo. To examine this possibility, we have investigated the subcellular localization of AQP-5 in rat parotid and submandibular glands fixed in vivo under conditions of stimulated or inhibited salivary secretion. Immunofluorescence and immunoelectron microscopy was used to determine the subcellular distribution of AQP-5 in control conditions following the stimulation of secretion with pilocarpine (a muscarinic agonist) or epinephrine (an alpha-adrenoceptor agonist) or during inhibition of basal secretion with atropine (a muscarinic antagonist) or phentolamine (an alpha-adrenoceptor antagonist). Under control conditions, >90% of AQP-5 was associated with the apical plasma membrane of acinar and intercalated duct cells, with only rare gold particles associated with intracellular membrane domains. Pilocarpine treatment dramatically increased saliva production but had no discernible effect on AQP-5 distribution. However, the increased salivary secretion was associated with luminal dilation and the appearance of a markedly punctate AQP-5 labeling pattern due to clustering of AQP-5 at the microvilli (especially evident in the parotid gland) after 10 min of drug injection. No changes in the subcellular localization of AQP-5 were seen in response to epinephrine, atropine, or phentolamine treatment compared with control tissues. Thus AQP-5 is localized predominantly in the apical plasma membrane under control conditions, and neither the onset nor the cessation of secretion is associated in vivo with any significant short-term translocation of AQP-5 between intracellular structures and the apical plasma membrane.     

Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy

Light-microscopy showed parotid serous acinar cells to contain neutral mucin, serous and mucous acinar cells of submandibular gland and intercalary ductal cells of both glands to contain acid and neutral mucins, and cells of striated ducts and excretory ducts to contain neutral mucin. Mucins were demonstrated ultrastructurally in a portion of the components of secretory granules of acinar cells and intercalary ductal cells, and in secretory granules of striated and excretory ductal cells. The mucins were all stained by techniques that reveal 1,2-glycols. Secretory granules of submandibular mucous and serous acinar cells and intercalary ductal cells were stained variably by the low iron-diamine technique for acid mucin, and those of mucous acinar cells by the high iron-diamine technique for sulphomucins mucin and possibly consisted of protein. The results suggest that one type of cell may be able to produce a range of secretory products and to package them variously into secretory granules.     

Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.     

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.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.     

Two-photon microscopic analysis of acetylcholine-induced mucus secretion in guinea pig nasal glands

The spatiotemporal changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) as well as fluid secretion and exocytosis induced by acetylcholine (ACh) in intact acini of guinea pig nasal glands were investigated by two-photon excitation imaging. Cross-sectional images of acini loaded with the fluorescent Ca(2+) indicator fura-2 revealed that the ACh-evoked increase in [Ca(2+)](i) was immediate and spread from the apical region (the secretory pole) of acinar cells to the basal region. Immersion of acini in a solution containing a fluorescent polar tracer, sulforhodamine B (SRB), revealed that fluid secretion, detected as a rapid disappearance of SRB fluorescence from the extracellular space, occurred exclusively in the luminal region and was accompanied by a reduction in acinar cell volume. Individual exocytic events were also visualized with SRB as the formation of Omega-shaped profiles at the apical membrane. In contrast to the rapidity of fluid secretion, exocytosis of secretory granules occurred with a delay of approximately 70s relative to the increase in [Ca(2+)](i). Exocytic events also occurred deep within the cytoplasm in a sequential manner with the latency of secondary exocytosis being greatly reduced compared with that of primary exocytosis. The delay in sequential compound exocytosis relative to fluid secretion may be important for release of the viscous contents of secretory granules into the nasal cavity.