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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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Regulation of exocytosis by cAMP and PKA. (A) Stimulation by different agents known to raise intracellular cAMP. Cells were treated with forskolin (20 μM), epinephrine (1 μM), VIP (1 μM), or 8-Br-cAMP (1 mM) for 3 min. Relative exocytosis is the ratio of exocytosis after and before the treatment. One group of cells (Forskolin + BAPTA) was pretreated with 50 μM BAPTA-AM for 1 h. (B) Evidence that PKA is needed. After the rate of exocytosis was measured in control saline solution, the PKA-specific inhibitor, Rp-8-Br-cAMPS, was applied to the bath for 5 min at an estimated final concentration of 2 mM. The rate of exocytosis in Rp-8Br-cAMPS was measured during the last 2 min of application of the inhibitor. Then, forskolin (∼20 μM) was added to the bath (Rp-8Br-cAMPS + Forskolin). In both cases, the rate of exocytosis was compared with that in control condition. With other sets of cells, the PKA-specific activator, Sp-8-Br-cAMPS, was applied to bath (Sp-8Br-cAMPS, ∼2 mM). The value for forskolin alone is same as in A. Cells were loaded with either dopamine or serotonin.
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Figure 6: Regulation of exocytosis by cAMP and PKA. (A) Stimulation by different agents known to raise intracellular cAMP. Cells were treated with forskolin (20 μM), epinephrine (1 μM), VIP (1 μM), or 8-Br-cAMP (1 mM) for 3 min. Relative exocytosis is the ratio of exocytosis after and before the treatment. One group of cells (Forskolin + BAPTA) was pretreated with 50 μM BAPTA-AM for 1 h. (B) Evidence that PKA is needed. After the rate of exocytosis was measured in control saline solution, the PKA-specific inhibitor, Rp-8-Br-cAMPS, was applied to the bath for 5 min at an estimated final concentration of 2 mM. The rate of exocytosis in Rp-8Br-cAMPS was measured during the last 2 min of application of the inhibitor. Then, forskolin (∼20 μM) was added to the bath (Rp-8Br-cAMPS + Forskolin). In both cases, the rate of exocytosis was compared with that in control condition. With other sets of cells, the PKA-specific activator, Sp-8-Br-cAMPS, was applied to bath (Sp-8Br-cAMPS, ∼2 mM). The value for forskolin alone is same as in A. Cells were loaded with either dopamine or serotonin.

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)

Regulation of exocytosis by cAMP and PKA. (A) Stimulation by different agents known to raise intracellular cAMP. Cells were treated with forskolin (20 μM), epinephrine (1 μM), VIP (1 μM), or 8-Br-cAMP (1 mM) for 3 min. Relative exocytosis is the ratio of exocytosis after and before the treatment. One group of cells (Forskolin + BAPTA) was pretreated with 50 μM BAPTA-AM for 1 h. (B) Evidence that PKA is needed. After the rate of exocytosis was measured in control saline solution, the PKA-specific inhibitor, Rp-8-Br-cAMPS, was applied to the bath for 5 min at an estimated final concentration of 2 mM. The rate of exocytosis in Rp-8Br-cAMPS was measured during the last 2 min of application of the inhibitor. Then, forskolin (∼20 μM) was added to the bath (Rp-8Br-cAMPS + Forskolin). In both cases, the rate of exocytosis was compared with that in control condition. With other sets of cells, the PKA-specific activator, Sp-8-Br-cAMPS, was applied to bath (Sp-8Br-cAMPS, ∼2 mM). The value for forskolin alone is same as in A. Cells were loaded with either dopamine or serotonin.
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Figure 6: Regulation of exocytosis by cAMP and PKA. (A) Stimulation by different agents known to raise intracellular cAMP. Cells were treated with forskolin (20 μM), epinephrine (1 μM), VIP (1 μM), or 8-Br-cAMP (1 mM) for 3 min. Relative exocytosis is the ratio of exocytosis after and before the treatment. One group of cells (Forskolin + BAPTA) was pretreated with 50 μM BAPTA-AM for 1 h. (B) Evidence that PKA is needed. After the rate of exocytosis was measured in control saline solution, the PKA-specific inhibitor, Rp-8-Br-cAMPS, was applied to the bath for 5 min at an estimated final concentration of 2 mM. The rate of exocytosis in Rp-8Br-cAMPS was measured during the last 2 min of application of the inhibitor. Then, forskolin (∼20 μM) was added to the bath (Rp-8Br-cAMPS + Forskolin). In both cases, the rate of exocytosis was compared with that in control condition. With other sets of cells, the PKA-specific activator, Sp-8-Br-cAMPS, was applied to bath (Sp-8Br-cAMPS, ∼2 mM). The value for forskolin alone is same as in A. Cells were loaded with either dopamine or serotonin.
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