Akihisa Segawa

Exocytosis in human salivary glands visualized by high-resolution scanning electron microscopy

Abstract The luminal membrane of salivary acinar cells creates a specialized cell surface area that accepts exocytosis and undergoes dynamic changes during secretion. These changes were visualized three-dimensionally from both the inside and outside of the cell in human parotid and submandibular glands, by application of in vitro secretory stimulation and then of OsO4 maceration to remove cytoplasmic organelles by varying degrees. In control glands treated without secretagogues, the luminal surface of serous acinar cells bore well-developed microvilli with only an occasional incidence of exocytotic profiles. Following treatment with the β-adrenergic agonist, isoproterenol, considerable shortening and loss of microvilli occurred along the luminal membrane where, on its cytoplasmic side, many protuberances of sizes similar to or smaller than those of single secretory granules (∼1 μm in diameter) appeared. The cytoplasmic surface of these protuberances exhibited small vesicles (∼100–150 nm in diameter) that, by transmission electron microscopy, were shown to be coated pits or vesicles present on or around the exocytosed granule membranes. Treatment of tissues with the muscarinic agonist carbachol also caused a decrease of microvilli and the appearance of protrusions at the luminal membrane. However, unlike isoproterenol treatment, many of these protrusions were devoid of small pits or vesicles and were much larger than a single secretory granule. These results indicate that (1) secretory stimulation causes the dynamic transformation of microvilli at the luminal membrane, where granule docking and membrane fusion take place, and (2) after fusion, the exocytosed membranes are processed differently, by coated pit/vesicle mediated or non-mediated mechanisms, according to the autonomic receptor control.

Cyclic AMP has distinct effects from Ca(2+) in evoking priming and fusion/exocytosis in parotid amylase secretion.

Abstract. Rat parotid acinar cells were perfused in small quartz columns to examine the role of cAMP and Ca2+ in the priming and fusion/exocytosis of amylase secretion. Carbachol (CCh) evoked a biphasic response of amylase secretion with an initial rapidly occurring large peak and a subsequent sustained plateau. Isoproterenol produced slowly increasing amylase secretion that reached the plateau greater than that induced by CCh. Combined stimulation with isoproterenol and CCh greatly potentiated amylase secretion. The rise and decay of amylase secretion induced by the combined stimulation was similar to those induced by CCh but not by isoproterenol, suggesting that the potentiation is caused by isoproterenol-induced modification of the CCh effect. Concentration-dependent responses of CCh-induced amylase secretion with and without isoproterenol showed that isoproterenol greatly enhances both the sensitivity and maximum effect of CCh. Similar potentiation was observed when the Ca2+ effect was directly examined in cells permeabilized to Ca2+ with ionomycin instead of CCh. In a Ca2+-free medium, CCh evoked an initial peak but did not produce a sustained plateau. Isoproterenol did not enhance the effect of CCh on [Ca2+]i. 2,4-Dintrophenol and carbonyl cyanide m-chlorophenyl hydrazone did not decrease the CCh-induced initial peak of amylase secretion but markedly decreased the sustained responses induced by isoproterenol and CCh. These results suggest that CCh, via Ca2+, has two distinct effects on amylase secretion: triggering of fusion/exocytosis and the priming of secretory granules. Isoproterenol, via cyclic AMP, also has two distinct effects: direct stimulation of priming and enhancement of the sensitivity to the Ca2+ effects. Thus, isoproterenol stimulates amylase secretion by increasing the primed pools of secretory granules, whereas CCh increases the flux of secretory granules into/from the primed pools, which is greatly enhanced by isoproterenol.

Relationship between amylase and fluid secretion in the isolated perfused whole parotid gland of the rat.

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly at 40-120 microliter/g-min (average plateau was 60 microliter/g-min), whereas amylase secretion exhibited an initial peak (10 mg maltose/30 s per g wet w. of the gland), followed by a rapid decrease to reach a plateau level of 1 mg maltose/30 s later than 1.5-2 min. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion to 15 mg maltose/30 s accompanied by the increase in oxygen consumption. However, the fluid secretion exhibited a rather gradual decrease. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed -exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. During washout of secretagogues, lysosomal digestion of excess membrane took place.

Carbachol-induced [Ca2+]i increase, but not activation of protein kinase C, stimulates exocytosis in rat parotid acini

A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)‐induced amylase secretion. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose‐response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i). The CCh‐induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3‐sensitive stores was relatively ATP independent. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca2+‐free medium evoked a second rapid and large peak of amylase secretion. Phorbol 12,13‐dibutyrate (PDBu) potentiated the CCh‐induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh‐induced [Ca2+]i changes. PKC inhibitors such as Ro 31–8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone. These results suggest that a CCh‐induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP‐dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh‐induced sustained plateau of amylase secretion. PDBu‐induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.

HIGH LEVELS OF GM1-GANGLIOSIDE AND GM1-GANGLIOSIDE β-GALACTOSIDASE IN THE PAROTID GLAND: A New Model for Secretory Mechanisms of the Parotid Gland

A new model for the subcellular basis of parotid secretion is presented in this article. GM(1)-ganglioside, typically found in neural tissues, is shown to be abundant in the parotid gland. This ganglioside may play a central role in membrane turnover mechanisms underlying exocytosis/endocytosis in its role as a promoter of membrane fusion or a fusogen. The lysosome and lysosomal hydrolases also play a central role in this model in catabolism of GM(1)-ganglioside. Consequently, high levels of the lysosomal hydrolase acidic beta-galactosidase are demonstrated in the salivary gland. GM(1)-gangliosidosis of the parotid glands, as described in mice, appears to be the first single-gene heritable disease found so far in the salivary glands.