Cholinergic Stimulation of Lacrimal Acinar Cells Promotes Redistribution of Membrane-associated Kinesin and the Secretory Protein, β-hexosaminidase, and Increases Kinesin Motor Activity

Hamm‐Alvarez, Sarah F.; Costa, SR Da; Yang, Tao; Wei, Xiaojing; Gierow, J. Peter; Mircheff, Austin K.

doi:10.1006/exer.1996.0198

Cited by 46 publications

(51 citation statements)

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“…Lacrimal gland acinar cells were isolated and cultured for 2-3 days as described previously. [21][22][23] …”

Section: Cell Isolation and Culturementioning

confidence: 99%

“…The cell pellet was then exposed to a cocktail of collagenase, hyaluronidase and DNAse 21,22 at 371C for 10 min, cells were again rinsed with DPBS and collected by centrifugation as described above. Cells were resuspended in DPBS, and GFP-positive cells were quantified by a Becton Dickinson FAC-Scant analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) using a 15 mW air-cooled argon laser set at 488 nm and recorded with a 530 nm emission filter in the FL1 emission channel.…”

Section: Flow Cytometry Analysismentioning

confidence: 99%

“…After washing in cold DPBS, acini were resuspended in 1 ml PMEE buffer (35 mM PIPES, 5 mM MgSO 4 , 1 mM EGTA, 0.5 mM EDTA, pH 7.4) containing protease inhibitors as described. 22 Cells were lysed by 20 passages through a 20-gauge syringe needle followed by 40 passages through a Balch cell press (H&Y Enterprises, Redwood City, CA, USA). The lysate was centrifuged for 10 min at 10 000 rpm in an Eppendorf 5415 C centrifuge at 41C.…”

Section: Membrane Fractionationmentioning

confidence: 99%

“…As an initial step toward this goal, we investigated the effect of replication-defective adenovirus serotype 5 (Ad5) containing either green fluorescent protein (GFP) or b-galactosidase (LacZ) on the organization and function of the lacrimal acinar secretory pathway in reconstituted acinus-like structures formed in vitro from acinar cells from rabbit lacrimal gland. [21][22][23] At conditions associated with high-efficiency transduction (480%), Ad elicited specific changes in the organization and function of the stimulated secretory pathway, changes that could be largely recapitulated by ultraviolet light (UV)-inactivated Ad. These findings suggest that ocular gene therapy utilizing Ad or Adderived products may alter secretory membrane-trafficking functions in epithelial cells of the ocular surface.…”

Section: Introductionmentioning

confidence: 99%

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Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

et al. 2004

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Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in 480% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UVinactivated Ad, carbachol-stimulated release of bulk protein and b-hexosaminidase were significantly (Pp0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

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“…Lacrimal gland acinar cells were isolated and cultured for 2-3 days as described previously. [21][22][23] …”

Section: Cell Isolation and Culturementioning

confidence: 99%

Section: Flow Cytometry Analysismentioning

confidence: 99%

Section: Membrane Fractionationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

et al. 2004

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“…Changes in endosomal\lysosomal markers and motors in MT-binding, ATP-release membranes were our principal focus ; the amounts of each marker in this fraction were calculated as a percentage of the recovery of marker across all three membrane pools before comparison across treatments. Membrane-associated dynein and kinesin were quantified by Western blotting and densitometry, whereas β-hexosaminidase and acid phosphatase activities were measured as described previously [18,19].…”

Section: Mt-affinity Purification Of Membranesmentioning

confidence: 99%

Increased protein phosphorylation of cytoplasmic dynein results in impaired motor function

Runnegar

Wei

1999

Biochemical Journal

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Inhibition of serine/threonine protein phosphatases in rat hepatocytes by okadaic acid and microcystin increased the phosphorylation of several components of the cytoplasmic dynein complex. UV light/vanadate cleavage and Western blot analysis revealed that two of these components with molecular masses of approx. 400 kDa and 74 kDa were dynein heavy- and intermediate-chains respectively. This increased phosphorylation resulted in inhibition of dynein ATPase activity, and reduced motor-dependent avidity of endosomal/lysosomal membranes for microtubules.

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Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface

et al. 1998

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CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane-endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjögren's syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjögren's autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses.

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Cholinergic Stimulation of Lacrimal Acinar Cells Promotes Redistribution of Membrane-associated Kinesin and the Secretory Protein, β-hexosaminidase, and Increases Kinesin Motor Activity

Cited by 46 publications

References 0 publications

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

Increased protein phosphorylation of cytoplasmic dynein results in impaired motor function

Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface

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