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Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells.

Koh DS, Moody MW, Nguyen TD, Hille B - J. Gen. Physiol. (2000)

Bottom Line: The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis.Thus, PKA is a downstream effector of cAMP.The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290, USA.

ABSTRACT
We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.

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(A) Complex exocytosis. Amperometric recording from a cell loaded with dopamine. Cells were treated with 1 mM 8-Br-cAMP as indicated by a bar. A quantal event (*) and a large and long-lasting complex event (**) are shown on an expanded time scale. The complex event contained 131× more charge than the average quantal events in this recording. One complex event was observed in 109 quantal events in this experiment. Filter frequency: 200 Hz. (B) A similar experiment to A. The cell was treated with thapsigargin 10 min before the start of recording. The marked events contained 130 (*) and 101 (**) times more charge than the average quantal event in the recording. Two complex events were observed in 538 quantal events in this experiment. Filter frequency: 100 Hz.
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Figure 11: (A) Complex exocytosis. Amperometric recording from a cell loaded with dopamine. Cells were treated with 1 mM 8-Br-cAMP as indicated by a bar. A quantal event (*) and a large and long-lasting complex event (**) are shown on an expanded time scale. The complex event contained 131× more charge than the average quantal events in this recording. One complex event was observed in 109 quantal events in this experiment. Filter frequency: 200 Hz. (B) A similar experiment to A. The cell was treated with thapsigargin 10 min before the start of recording. The marked events contained 130 (*) and 101 (**) times more charge than the average quantal event in the recording. Two complex events were observed in 538 quantal events in this experiment. Filter frequency: 100 Hz.

Mentions: We occasionally observed large, long-lasting amperometric events. These complex events were characterized by multiple overlapping peaks, suggesting compound exocytosis with overlapping fusion of a large number of vesicles (Fig. 11). As an empirical criterion to detect such events, we used a sliding window of 50 adjacent sample points. A large event was scored if the total charge in this interval exceeded 20× the average charge of a single quantal event. The window was moved by 25 points each time to ensure the detection of the complex events. By this criterion, there were 12 giant events out of 2,714 amperometric events (one per 226 events) in seven experiments. The occurrence may be overestimated, as only recordings containing at least one obvious complex event were included in this analysis. The average charge of such complex amperometric events was 99 ± 25 (n = 7) times that of the quantal events. Such events were seen both during stimulated release and during spontaneous release in unstimulated cells.


Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells.

Koh DS, Moody MW, Nguyen TD, Hille B - J. Gen. Physiol. (2000)

(A) Complex exocytosis. Amperometric recording from a cell loaded with dopamine. Cells were treated with 1 mM 8-Br-cAMP as indicated by a bar. A quantal event (*) and a large and long-lasting complex event (**) are shown on an expanded time scale. The complex event contained 131× more charge than the average quantal events in this recording. One complex event was observed in 109 quantal events in this experiment. Filter frequency: 200 Hz. (B) A similar experiment to A. The cell was treated with thapsigargin 10 min before the start of recording. The marked events contained 130 (*) and 101 (**) times more charge than the average quantal event in the recording. Two complex events were observed in 538 quantal events in this experiment. Filter frequency: 100 Hz.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230622&req=5

Figure 11: (A) Complex exocytosis. Amperometric recording from a cell loaded with dopamine. Cells were treated with 1 mM 8-Br-cAMP as indicated by a bar. A quantal event (*) and a large and long-lasting complex event (**) are shown on an expanded time scale. The complex event contained 131× more charge than the average quantal events in this recording. One complex event was observed in 109 quantal events in this experiment. Filter frequency: 200 Hz. (B) A similar experiment to A. The cell was treated with thapsigargin 10 min before the start of recording. The marked events contained 130 (*) and 101 (**) times more charge than the average quantal event in the recording. Two complex events were observed in 538 quantal events in this experiment. Filter frequency: 100 Hz.
Mentions: We occasionally observed large, long-lasting amperometric events. These complex events were characterized by multiple overlapping peaks, suggesting compound exocytosis with overlapping fusion of a large number of vesicles (Fig. 11). As an empirical criterion to detect such events, we used a sliding window of 50 adjacent sample points. A large event was scored if the total charge in this interval exceeded 20× the average charge of a single quantal event. The window was moved by 25 points each time to ensure the detection of the complex events. By this criterion, there were 12 giant events out of 2,714 amperometric events (one per 226 events) in seven experiments. The occurrence may be overestimated, as only recordings containing at least one obvious complex event were included in this analysis. The average charge of such complex amperometric events was 99 ± 25 (n = 7) times that of the quantal events. Such events were seen both during stimulated release and during spontaneous release in unstimulated cells.

Bottom Line: The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis.Thus, PKA is a downstream effector of cAMP.The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290, USA.

ABSTRACT
We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.

Show MeSH
Related in: MedlinePlus