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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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Charge distributions of amperometric events in control and forskolin. Amperometric events were pooled from six consecutive experiments using the same electrode. To acquire enough events, cells were recorded for 5–10 min in control before the 5-min forskolin (20 μM) treatment. For forskolin, the 2-min analysis period started after 2 min and the mean rate was about 2.5× that in the control. 518 events larger than 1 fC were analyzed for each histogram. The smallest bars are single events.
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Figure 5: Charge distributions of amperometric events in control and forskolin. Amperometric events were pooled from six consecutive experiments using the same electrode. To acquire enough events, cells were recorded for 5–10 min in control before the 5-min forskolin (20 μM) treatment. For forskolin, the 2-min analysis period started after 2 min and the mean rate was about 2.5× that in the control. 518 events larger than 1 fC were analyzed for each histogram. The smallest bars are single events.

Mentions: Amperometric recordings were semiautomatically analyzed using software written in Igor (WaveMetrics). Some recordings with a small number of amperometric signals were plotted on a fast chart recorder and events were counted manually. The rate of exocytosis was defined as the number of amperometric spikes per 30-s time bin. To evaluate relative exocytosis, the rates of exocytosis in control and test conditions were averaged for 2 min. As the maximal exocytosis is reached at different times in different test solutions, the 2-min analysis period was taken after various time delays: 3 min for forskolin or PMA on 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)–loaded cells (see Fig. 4, and Fig. 6Fig. 7Fig. 8), Rp-8-Br-cAMPS (see Fig. 6) or BIS (see Fig. 8) treatments, 2 min for forskolin, PMA or Sp-8-Br-cAMPS on cells not loaded with BAPTA (see Fig. 5, Fig. 6, and Fig. 8), and 1 min for 2 mM Ca2+ on ionomycin- (see Fig. 2) and hyperosmotic solution-treated (see Fig. 10) cells. During treatments with hyperosmotic solution, only the last minute of the recordings was analyzed. Relative exocytosis was defined by the ratio of exocytosis rates after and before treatments.


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)

Charge distributions of amperometric events in control and forskolin. Amperometric events were pooled from six consecutive experiments using the same electrode. To acquire enough events, cells were recorded for 5–10 min in control before the 5-min forskolin (20 μM) treatment. For forskolin, the 2-min analysis period started after 2 min and the mean rate was about 2.5× that in the control. 518 events larger than 1 fC were analyzed for each histogram. The smallest bars are single events.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Charge distributions of amperometric events in control and forskolin. Amperometric events were pooled from six consecutive experiments using the same electrode. To acquire enough events, cells were recorded for 5–10 min in control before the 5-min forskolin (20 μM) treatment. For forskolin, the 2-min analysis period started after 2 min and the mean rate was about 2.5× that in the control. 518 events larger than 1 fC were analyzed for each histogram. The smallest bars are single events.
Mentions: Amperometric recordings were semiautomatically analyzed using software written in Igor (WaveMetrics). Some recordings with a small number of amperometric signals were plotted on a fast chart recorder and events were counted manually. The rate of exocytosis was defined as the number of amperometric spikes per 30-s time bin. To evaluate relative exocytosis, the rates of exocytosis in control and test conditions were averaged for 2 min. As the maximal exocytosis is reached at different times in different test solutions, the 2-min analysis period was taken after various time delays: 3 min for forskolin or PMA on 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)–loaded cells (see Fig. 4, and Fig. 6Fig. 7Fig. 8), Rp-8-Br-cAMPS (see Fig. 6) or BIS (see Fig. 8) treatments, 2 min for forskolin, PMA or Sp-8-Br-cAMPS on cells not loaded with BAPTA (see Fig. 5, Fig. 6, and Fig. 8), and 1 min for 2 mM Ca2+ on ionomycin- (see Fig. 2) and hyperosmotic solution-treated (see Fig. 10) cells. During treatments with hyperosmotic solution, only the last minute of the recordings was analyzed. Relative exocytosis was defined by the ratio of exocytosis rates after and before treatments.

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