cAMP and Ca(2+)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton

The potential involvement of actin and fodrin (brain spectrin) in secretory events has been assessed in primary cultured guinea pig parotid acinar cells, using as a tool affinity purified anti-alpha-fodrin antibody, phalloidin, and immunofluorescence techniques. In resting parotid acinar cells fodrin and actin appeared as a continuous ring under the plasma membrane of most of the cells. Upon stimulation with secretagogues fodrin and actin labeling at the level of the plasma membrane disappeared almost completely. To establish a correlation between secretion and cytoskeletal changes at the individual cell level, anti-alpha-amylase-antibodies were used to label secreted amylase exposed at the surface of secreting cells. The number of cells expressing alpha-amylase on their surface followed bulk secretion of alpha-amylase. A strict correlation between secretion and alteration of the actin-fodrin labeling was observed at the individual cell level. The cytoskeletal changes occurred in parallel with secretion independently of the secretagogue used (carbamoylcholine in the presence of Ca2+, isoproterenol in presence or absence of Ca2+, forskolin, or dibutyryl-cyclic-AMP). The changes were reversible upon removal of the secretagogue. Since Ca2+, as well as cAMP-mediated secretion, was associated with the same kind of cytoskeletal changes, a reorganization of the cytoskeleton may play an essential part in regulated secretion.     

Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.     

PKA-mediated protein phosphorylation protects ezrin from calpain I cleavage

Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase (PKA) activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies demonstrate the functional relevance of PKA-mediated phosphorylation of ezrin in parietal cell secretion [R. Zhou, X. Cao, C. Watson, Y. Miao, Z. Guo, J.G. Forte, X. Yao, Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651]. Here we show that activation of PKA protects ezrin from calpain I-mediated proteolysis without alteration of calpain I activation and fodrin breakdown. To determine whether phosphorylation of Ser66 by PKA affects the insensitivity to the calpain I-mediated cleavage, recombinant proteins of ezrin, both wild type and S66A/D mutants, were incubated with the purified calpain I. Indeed, phosphorylation-like S66D mutant ezrin is resistant to calpain I-mediated proteolysis while wild type and S66A mutant were sensitive. In fact, expression of phosphorylation-like S66D, but not S66A, mutant in parietal cells confers its resistance to calpain I-mediated proteolysis. Taken together, these results indicate that phosphorylation of ezrin by PKA modulates its sensitivity to calpain I cleavage.     

The 1989 Upjohn Award lecture. Cellular and molecular mechanisms in hormone and neurotransmitter secretion

Studies on adrenal medulla have had an important influence on the development of a variety of biological concepts, not only within the area of endocrinology, but also in the areas of chemical neurotransmission and secretion in general. The adrenal medulla chromaffin cells are derived embryologically from the neural crest, sharing a common origin with sympathetic neurons and common subcellular features with many endocrine cells. One such feature is the storage of secretory products in membrane-bound organelles, the secretory granules. Secretory cells with these characteristics have been named paraneurons, a term that embraces cells generally and traditionally not considered as neurons, and yet should be regarded as relatives of neurons on the basis of their structure, function, and metabolism. Many of the studies carried out in the past to understand the secretory process have employed perfused adrenal glands. Although this technique has provided very useful information regarding secretion, it did not allow the study of the cellular events involved in the secretory process. To obtain further information on cell secretion, several laboratories including our own have published methods for the isolation and culture of chromaffin cells. The cultured chromaffin cells have shown themselves to be one of the most useful systems developed for the study of the neuroendocrine functions of paraneurons. Studies on cultured chromaffin cells have provided important information on secretory cell cytoskeleton: a group of proteins, some of them previously known from studies on muscle, which form a cytoplasmic network in all non-muscle cells including secretory cells. Immunohistochemical studies have shown at least three types of filament systems: microfilaments, microtubules, and intermediate filaments. In addition, a large variety of cytoskeleton-associated proteins have been characterized. Chromaffin cells are among those non-muscle cells from which cytoskeleton proteins have been isolated and characterized. Owing to similarities between "stimulus-secretion coupling" and "excitation-contraction coupling" in muscle, it has been proposed that the secretory process might be mediated by contractile elements either associated with secretory vesicles or present elsewhere in the secretory cell. Cytoskeletal proteins (actin, myosin, alpha-actinin, fodrin, tubulin, and neurofilament subunits) and their regulatory proteins (calmodulin, gelsolin) have been isolated from chromaffin cells and characterized. Their physiochemical proteins have been studied and their cellular localizations have been revealed by biochemical, immunocytochemical, and ultrastructural techniques. alpha-Actinin and fodrin are components of chromaffin granule membranes and some of the cell actin co-purified with secretory granules. Actin forms a network of microfilaments in the subplasmalemma region.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.     

Neuronal fodrin proteolysis occurs independently of excitatory amino acid-induced neurotoxicity.

In cultured cerebellar granule cells, the total amount of fodrin alpha subunit increased 3-fold between 0 and 10 days in vitro and fodrin mRNA increased 5-fold. The exposure of cerebellar neurons to NMDA induced the accumulation of a 150 kd proteolytic fragment of fodrin. The NMDA-induced breakdown of fodrin was time-, concentration-, and Ca2(+)-dependent and was inhibited by APV, Mg2+, or the calpain I inhibitor N-acetyl-Leu-Leu-norleucinal. Kainate caused fodrin proteolysis through indirect activation of NMDA receptors. Quisqualate was ineffective. The NMDA-induced degradation of fodrin occurred under conditions that did not cause degeneration of cultured cerebellar neurons. These results show that Ca2+/calpain I-dependent proteolysis of fodrin is selectively associated with NMDA receptor activation; however, fodrin proteolysis per se does not play a causal role in NMDA-induced toxicity in cerebellar granule cells.     

Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin

We examined the distribution of fodrin and cytochrome b561 in secretion-induced rat chromaffin cells (epinephrine cells) by immunofluorescence and immunoelectron microscopy. Fasted rats injected with a large dose of insulin were perfusion-fixed and frozen sections of the adrenal medulla were immunolabeled. Fodrin, a peripheral membrane protein, was distributed only in the cell periphery in control cells, but was observed in the cell interior after the insulin treatment; many of the markers were found around small vesicles, 50-200 nm in diameter, and large vacuoles, more than 500 nm in diameter. On the other hand, cytochrome b561, an integral membrane protein, was seen only in the chromaffin granules in control cells, and appeared in small vesicles in the stimulated cells but not in large vacuoles. By double immunolabeling it was shown that cytochrome b561 coexisted with fodrin in the small vesicles. The coexistence of the two proteins was confirmed by the labeling of subcellular particles immunoadsorbed from the insulin-treated adrenal medulla homogenate; vesicles immunoisolated with anti-fodrin antibody on polyacrylamide beads were positively immunolabeled with anti-cytochrome b561 antibody. The present results show that during massive secretion fodrin is taken into the cell interior by vesicles, which may be a mechanism that retrieves the secretory granule membrane from the cell surface.     

Immunolocalization of a spectrin-like protein (fodrin) in pancreatic acinar cells

A spectrin-like protein (fodrin) was localized in porcine pancreas using an immunoperoxidase procedure with antibodies raised against erythrocyte spectrin. Fodrin was primarily associated with the cell plasma membrane although some was also detectable in the cytoplasm of the acinar cells. The membrane labelling of the acinar cells was uneven such that the lateral and basal membranes were strongly labelled by anti-spectrin antibodies whereas the apical membranes were poorly labelled. The implications of the results to secretion and to the occurrence of specific membrane domains are discussed.     

A putative role for calpain in demyelination associated with optic neuritis

Calcium activated neutral proteinase (calpain) is an endopeptidase present in the central nervous system which degrades myelin proteins. To examine the role of calpain in demyelination associated with optic neuritis, immunocytochemical expression of calpain was evaluated in Lewis rats with experimental optic neuritis. Calpain expression was increased in activated microglia, infiltrating macrophages, activated T cells, and reactive astrocytes in experimental optic neuritis compared to controls. Calpain activity and translational expression were also examined by Western blotting studies measuring the extent of myelin protein degradation, calpain-specific fodrin proteolysis, axonal neurofilament degradation, and calpain proenzyme content. Results showed myelin associated glycoprotein and 68 kD neurofilament protein levels were significantly decreased while calpain translational expression and calpain-autolyzed fodrin levels were significantly increased in experimental optic neuritis compared to controls. Thus, increased activity and translational expression of calpain in optic neuritis may be integral to the pathogenesis of this disorder.     

Suppression of carbachol-induced oscillatory Cl- secretion by forskolin in rat parotid and submandibular acinar cells

Sympathetic stimulation induces weak salivation compared with parasympathetic stimulation. To clarify this phenomenon in salivary glands, we investigated cAMP-induced modulation of Ca(2+)-activated Cl(-) secretion from rat parotid and submandibular acinar cells because fluid secretion from salivary glands depends on the Cl(-) secretion. Carbachol (Cch), a Ca(2+)-increasing agent, induced hyperpolarization of the cells with oscillatory depolarization in the current clamp mode of the gramicidin-perforated patch recording. In the voltage clamp mode at -80 mV, Cch induced a bumetanide-sensitive oscillatory inward current, which was larger in rat submandibular acinar cells than in parotid acinar cells. Forskolin and IBMX, cAMP-increasing agents, did not induce any marked current, but they evoked a small nonoscillatory inward current in the presence of Cch and suppressed the Cch-induced oscillatory inward current in all parotid acinar cells and half (56%) of submandibular acinar cells. In the current clamp mode, forskolin + IBMX evoked a small nonoscillatory depolarization in the presence of Cch and reduced the amplitude of Cch-induced oscillatory depolarization in both acinar cells. The oscillatory inward current estimated at the depolarized membrane potential was suppressed by forskolin + IBMX. These results indicate that cAMP suppresses Ca(2+)-activated oscillatory Cl(-) secretion of parotid and submandibular acinar cells at -80 mV and possibly at the membrane potential during Cch stimulation. The suppression may result in the weak salivation induced by sympathetic stimulation.     

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.     

Physiological roles of the intermediate conductance, Ca2+-activated potassium channel Kcnn4

Three broad classes of Ca(2+)-activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively. SK channels are likely encoded by three genes, Kcnn1-3, whereas IK and most BK channels are most likely products of the Kcnn4 and Slo (Kcnma1) genes, respectively. IK channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands. IK channels likely underlie the K(+) permeability in red blood cells that is associated with water loss, which is a contributing factor in the pathophysiology of sickle cell disease. IK channels are also involved in the activation of T lymphocytes. The fluid-secreting acinar cells of the parotid gland express both IK and BK channels, raising questions about their particular respective roles. To test the physiological roles of channels encoded by the Kcnn4 gene, we constructed a mouse deficient in its expression. Kcnn4 null mice were of normal appearance and fertility, their parotid acinar cells expressed no IK channels, and their red blood cells lost K(+) permeability. The volume regulation of T lymphocytes and erythrocytes was severely impaired in Kcnn4 null mice but was normal in parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from parotid glands was normal. These results confirm that IK channels in red blood cells, T lymphocytes, and parotid acinar cells are indeed encoded by the Kcnn4 gene. The role of these channels in water movement and the subsequent volume changes in red blood cells and T lymphocytes is also confirmed. Surprisingly, Kcnn4 channels appear to play no required role in fluid secretion and regulatory volume decrease in the parotid gland.     

Degradation of fodrin by m-calpain in fibroblasts adhering to fibrillar collagen I gel

When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas micro-calpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, alpha-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel.     

Prion Pathogenesis Is Faithfully Reproduced in Cerebellar Organotypic Slice Cultures

Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.     

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.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

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.     

Localization of 5′-nucleotidase activity in the parotid acinar cells of a rat treated with isoproterenol

Summary Cytochemical localization of 5- nucleotidase (AMPase) has been investigated in the parotid acinar cells of rats at various stages of exocytic secretion induced by an administration of isoproterenol (IPR).In the resting stage, the acinar cells show AMPase activity located on the baso-lateral and luminal plasmalemma, and in the earliest secretory stage the luminal plasma membranes are devoid of the enzymatic activity. However, these particular regions exhibit AMPase activity during the advanced stages of secretion, and the AMPase positive membranes become absorbed into the cytoplasm by endocytic activity. The absorbed membrane components then seem to be degraded by the action of lysosomes.The intracellular fate of the endocytic vacuoles has been examined by the aid of ferritin particles introduced retrogradely through ductal lumina. Ferritin containing vacuoles are distributed in the cytoplasm, and these droplets change into secondary lysosomes. No tracer particles are recognized in the internal space of the Golgi lamella and its associated vesicles.The results suggested that in the exocytic secretion of parotid acinar cells, AMPase originating from plasma membrane intermingles with the membranes derived from secretion granules, and is translocated into cytoplasm by an endocytic mechanism. The internalized membrane components are, at least partly, degraded by lysosome action.     

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.