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The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells

Anna M. Castle^*, Amy Y. Huang^* and J. David Castle

Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908-0732, USA
^* These authors contributed equally to this work

View larger version (22K): [in a new window]   Fig. 1. Discharge of the minor regulated pathway precedes granule exocytosis. (A) Coomassie staining profiles for parotid secretory proteins released at early time points after pulse-chase biosynthetic labeling with [35S]amino acids upon stimulation of lobules by 10 µM isoproterenol. At each time point, the medium was removed and replaced with fresh medium containing freshly added stimulant. Amylase (Amy.), proline-rich protein (PRP), and parotid secretory protein (PSP) are identified. The composition of secretion elicited by 40 nM carbachol (CCh) and of the content of isolated secretory granules (Gr) is also shown. (B) Radiochemical composition of protein profiles illustrated in panel A. PRP contains no methionine or cysteine and is unlabeled. (C) Specific radioactivity of amylase for the samples shown in panels A and B obtained by normalizing the intensities of the amylase bands in B (quantified by phosphorimaging) to the amylase enzyme activity in each sample.

View larger version (19K): [in a new window]   Fig. 2. Characterization of secretion stimulated by different doses of carbachol. Parotid lobules were pulse labeled and chased for 180 minutes before stimulation and, at each timepoint, the medium was completely removed and replaced. (A) Time course of secretion of amylase enzyme upon stimulation with 40 nM CCh, 200 nM CCh and 2 µM CCh. (B) Specific radioactivity of amylase was calculated as in Fig. 1. (C) Left panel, radiochemical composition of secretion elicited by 2 µM CCh after 1 minute of stimulation. Right panel, coomassie-stained profiles for samples of secretion collected following stimulation with 40 nM (lanes 1,2) and 2 µM (lanes 3,4) CCh. Lanes 1 and 3 show profiles for the first 3 minutes of stimulation, lanes 2 and 4 show profiles from the 10-15 minute interval. For comparison, lane 5 shows a sample collected after 15 minutes of stimulation with 1 µM Iso. Data are from one of three experiments with the same outcome.

View larger version (29K): [in a new window]   Fig. 3. Potentiation of isoproterenol-stimulated amylase secretion by CCh. Parotid lobules were pulse-labeled and chased as in Fig. 1. (A) Secretion of amylase enzyme from samples stimulated with 40 nM CCh alone (CCh), 1 µM Iso (Iso), 40 nM CCh and 1 µM Iso in combination (CCh + Iso). A parallel sample was treated with 40 nM CCh and 1 µM Iso for 10 minutes and subsequently with 1 µM Iso alone (CCh take-away). At each timepoint the medium was removed in entirely and replaced with medium to which additives had been freshly added. (B) Specific radioactivity of amylase in samples stimulated with 1 µM Iso, 1 µM Iso and 40 nM CCh, or 1 µM Iso and 200 nM CCh during the first 10 minutes of stimulation. At each time point the medium was completely removed and replaced. (C) Effect of pre-stimulation with 40 nM CCh on initial secretion and specific radioactivity of amylase. Lobules were pulsed and chased as above. Amylase enzyme (left panel) and specific radioactivity of amylase (right panel) were determined in secretion from the first 1 minute of stimulation in samples treated with 1 µM Iso (open bar), 1 µM Iso and 40 nM CCh added simultaneously (hashed bar), and a sample pre-treated with 40 nM CCh 5 minutes prior to addition of 1 µM Iso plus 40 nM CCh (filled bar). Medium was completely replaced at the end of the pretreatment. (D) Time course of secretion of radiolabeled PSP as a marker of newly formed granules. The gels shown were those from the experiment shown in B.

View larger version (19K): [in a new window]   Fig. 4. Potentiation of isoproterenol-stimulated amylase secretion by BFA and by K+ depletion. Parotid lobules were pulse-labeled and chased as in Fig. 1. (A) Effects of BFA treatment on amylase secretion. Lobules were pretreated with 10 µg/ml BFA or carrier Me2SO for 30 minutes. Subsequently samples were treated with 10 µg/ml BFA alone (BFA), 1 µM Iso and carrier Me2SO (Iso) or 10 µg/ml BFA (Iso + BFA) and incubation was continued for 60 minutes with complete changes of medium every 10 minutes. A parallel sample was treated with 1 µM Iso and 10 µg/ml BFA and subsequently with 1 µM Iso alone (BFA take-away). The results shown are representative of four separate experiments. (B) Potentiation of isoproterenol-induced amylase secretion in K+-depleted medium. Samples were incubated in DMEM or K+-free DMEM (Na+ replacing K+) for 30 minutes before adding 1 µM isoproterenol. Subsequently, incubation was continued with complete changes of medium every 10 minutes. The results shown are representative of four separate experiments.

View larger version (135K): [in a new window]   Fig. 5. Localization of syntaxin 3 in unstimulated parotid tissue. (A,C,D) Immunofluorescence of cryosections of parotid tissue have been stained with anti-syntaxin 3 (red) and either BODIPY-phallacidin to mark the actin-rich terminal web or anti-DPPIV to mark the apical plasma membrane (green). All other fluorescent images are of a single 0.1 µM layer following digital deconvolution. Closed arrowheads identify some of the syntaxin 3 foci that are concentrated beneath (on the cytoplasmic side of) the actin band or the apical plasma membrane and are found where little or no luminal space is visible. Open arrrowheads identify some of the few syntaxin 3 foci that have relocated to the luminal side of the actin band or into the DPPIV band signifying association with the apical plasma membrane. Relocated foci are generally observed where the luminal space is evident. (B) Differential interference contrast (DIC) image paired with the image in A. Bar, 10 µm.

View larger version (138K): [in a new window]   Fig. 6. Relocation of syntaxin 3 in response to stimulation of minor regulated pathway. Parotid tissue was processed for immunofluorescence following a 3 minute treatment with 40 nM CCh or 1 µM Iso. (A,B,D) Immunofluorescent specimens were stained with syntaxin 3 (red) and counterstained with BODIPY-phallacidin (green) or DPPIV (green). (C) Comparison of staining of DPPIV (red) and BODIPY-phallacidin (green). Open arrowheads identify examples of syntaxin 3 foci that have relocated to the luminal side of the actin band or into the DPPIV band. Bar, 10 µm.

View larger version (157K): [in a new window]   Fig. 7. Evaluation of other vesicular trafficking proteins as possible marker of carriers of the minor regulated pathway. Parotid tissue was processed for immunofluorescence without (Ctl) or with (Cch) a 3 minute treatment with 40 nM CCh. Immunofluorescent specimens have been counterstained with BODIPY-phallacidin (green) for actin in each case. (A) VAMP2 (red) staining of control tissue; (B) VAMP2 (red) staining of stimulated tissue; (C) Rab11 (red) staining of control tissue; (D) Rab11 (red) staining of stimulated tissue. Solid arrowheads identify prospective subapical foci of VAMP2 and Rab 11, and open arrowheads identify some of the foci that have relocated to the luminal side of the actin band. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 8. Effect of BFA treatment or K+-depletion on localization of syntaxin 3. Parotid lobules were incubated for 15 minutes with 10 µg/ml BFA (A) or in K+-free medium (B) prior to fixation. Tissue sections were stained with syntaxin 3 (red) and counterstained with BODIPY-phallacidin (green). Open arrowheads point to examples of fluorescent foci that have relocated to the luminal aspect of the actin band. Bar, 10 µm.

View larger version (59K): [in a new window]   Fig. 9. Current model for the regulation of granule exocytosis in parotid acinar cells in which the minor regulated pathway provides granule docking/fusion sites. Abbreviations: CL, constitutive-like pathway; CV, condensing vacuole; IG, immature granule; MR, minor regulated pathway; SG, secretory granule. (A) Selective stimulation of the minor regulated pathway (red vesicles) by 40 nM CCh. Analogous selective stimulation occurs in response to <100 nM Iso (Castle and Castle, 1996). Discharge creates docking/fusion sites for secretory granules. (B) Sequential exocytosis of the minor regulated pathway and oldest secretion granules accumulated nearest to the apical surface in response to 2 µM CCh. (C) Sequential exocytosis of the minor regulated pathway and compound exocytosis leading to release of newly synthesized granules deep in the storage pool in response to 1 µM isoproterenol (Iso). (D) Secretory potentiation in response to 40 nM CCh and 1 µM isoproterenol resulting from multiple pathways of compound exocytosis at multiple docking/fusion sites created by the minor regulated pathway.

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