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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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Quantal secretory events detected in a dog pancreatic duct epithelial cell. (A) Representative amperometric signals. The cell was loaded in 70 mM serotonin solution and exocytotic events were monitored with a carbon-fiber electrode polarized at +600 mV in serotonin-free solution. (B) Quantal events from the same experiment plotted at higher time resolution. The arrows indicate “feet” preceding main spikes. The last event on the right is plotted on a different time scale. (C) Charge distribution histogram from the same experiment. Integrals of 105 individual amperometric spikes acquired without stimulation were analyzed. (D) Peak amplitude histogram of the same recording. Filter frequency: 200 Hz.
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Figure 1: Quantal secretory events detected in a dog pancreatic duct epithelial cell. (A) Representative amperometric signals. The cell was loaded in 70 mM serotonin solution and exocytotic events were monitored with a carbon-fiber electrode polarized at +600 mV in serotonin-free solution. (B) Quantal events from the same experiment plotted at higher time resolution. The arrows indicate “feet” preceding main spikes. The last event on the right is plotted on a different time scale. (C) Charge distribution histogram from the same experiment. Integrals of 105 individual amperometric spikes acquired without stimulation were analyzed. (D) Peak amplitude histogram of the same recording. Filter frequency: 200 Hz.

Mentions: We begin by describing the amperometric signature of exocytosis in epithelial cells for comparison with better-characterized systems. Touching a polarized carbon-fiber electrode gently to a single cell previously loaded in 70 mM dopamine or serotonin revealed spontaneous amperometric current spikes (Fig. 1A and Fig. B). They reflect rapid oxidation of many thousands of oxidizable molecules released abruptly at the cell surface. In tests on five different batches of cells, signals were not observed without preloading the amine, indicating that the spikes represented the exogenous oxidizable amine. It was necessary to incubate epithelial cells in the 70-mM loading medium for longer than 30 min to detect the oxidation currents. Attempting to load with 1.5 mM of dopamine or serotonin for 4 h elicited much smaller peaks (not shown). Rates of spontaneous exocytosis ranged from 2 to 53 events per min (mean: 17.2 ± 4.9 events/min, n = 11 cells). The rate typically decreased over 4–5 h after loading was stopped, presumably due to a loss of vesicular amines through spontaneous exocytosis and by leak of cytoplasmic amines to the culture medium.


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)

Quantal secretory events detected in a dog pancreatic duct epithelial cell. (A) Representative amperometric signals. The cell was loaded in 70 mM serotonin solution and exocytotic events were monitored with a carbon-fiber electrode polarized at +600 mV in serotonin-free solution. (B) Quantal events from the same experiment plotted at higher time resolution. The arrows indicate “feet” preceding main spikes. The last event on the right is plotted on a different time scale. (C) Charge distribution histogram from the same experiment. Integrals of 105 individual amperometric spikes acquired without stimulation were analyzed. (D) Peak amplitude histogram of the same recording. Filter frequency: 200 Hz.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230622&req=5

Figure 1: Quantal secretory events detected in a dog pancreatic duct epithelial cell. (A) Representative amperometric signals. The cell was loaded in 70 mM serotonin solution and exocytotic events were monitored with a carbon-fiber electrode polarized at +600 mV in serotonin-free solution. (B) Quantal events from the same experiment plotted at higher time resolution. The arrows indicate “feet” preceding main spikes. The last event on the right is plotted on a different time scale. (C) Charge distribution histogram from the same experiment. Integrals of 105 individual amperometric spikes acquired without stimulation were analyzed. (D) Peak amplitude histogram of the same recording. Filter frequency: 200 Hz.
Mentions: We begin by describing the amperometric signature of exocytosis in epithelial cells for comparison with better-characterized systems. Touching a polarized carbon-fiber electrode gently to a single cell previously loaded in 70 mM dopamine or serotonin revealed spontaneous amperometric current spikes (Fig. 1A and Fig. B). They reflect rapid oxidation of many thousands of oxidizable molecules released abruptly at the cell surface. In tests on five different batches of cells, signals were not observed without preloading the amine, indicating that the spikes represented the exogenous oxidizable amine. It was necessary to incubate epithelial cells in the 70-mM loading medium for longer than 30 min to detect the oxidation currents. Attempting to load with 1.5 mM of dopamine or serotonin for 4 h elicited much smaller peaks (not shown). Rates of spontaneous exocytosis ranged from 2 to 53 events per min (mean: 17.2 ± 4.9 events/min, n = 11 cells). The rate typically decreased over 4–5 h after loading was stopped, presumably due to a loss of vesicular amines through spontaneous exocytosis and by leak of cytoplasmic amines to the culture medium.

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