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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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Possible role of protein kinases in Ca2+-induced exocytosis. Bars represent relative exocytosis compared with either untreated control recording (closed bars) or recording obtained with the inhibitors H-89 or BIS (open bars). All statistical tests compare the data in the open bars with calcium treatments in uninhibited cells. (A) Exocytosis induced by small [Ca2+]i elevations. Relative rates of exocytosis induced by 300 μM Ca2+ alone (0.3Ca), or after incubation for 5 min with 10 μM H-89 (H-89), H-89 with 300 μM Ca2+ (H-89 + 0.3Ca), 500 nM BIS (BIS), or BIS with 300 μM Ca2+ (BIS + 0.3Ca). (B) Exocytosis induced by higher [Ca2+]i. Relative rates of exocytosis with 2 mM Ca2+ alone (2Ca), H-89 with 2 mM Ca2+ (H-89 + 2Ca), BIS with 2 mM Ca2+ (BIS + 2Ca), combined treatments of H-89 and BIS (H/BIS), or H-89 and BIS with 2 mM Ca2+ (H/BIS + 2Ca). The rate of exocytosis was measured for 3 min in controls and 5 min for inhibitors or inhibitors with Ca2+. Cells were loaded with dopamine.
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Figure 9: Possible role of protein kinases in Ca2+-induced exocytosis. Bars represent relative exocytosis compared with either untreated control recording (closed bars) or recording obtained with the inhibitors H-89 or BIS (open bars). All statistical tests compare the data in the open bars with calcium treatments in uninhibited cells. (A) Exocytosis induced by small [Ca2+]i elevations. Relative rates of exocytosis induced by 300 μM Ca2+ alone (0.3Ca), or after incubation for 5 min with 10 μM H-89 (H-89), H-89 with 300 μM Ca2+ (H-89 + 0.3Ca), 500 nM BIS (BIS), or BIS with 300 μM Ca2+ (BIS + 0.3Ca). (B) Exocytosis induced by higher [Ca2+]i. Relative rates of exocytosis with 2 mM Ca2+ alone (2Ca), H-89 with 2 mM Ca2+ (H-89 + 2Ca), BIS with 2 mM Ca2+ (BIS + 2Ca), combined treatments of H-89 and BIS (H/BIS), or H-89 and BIS with 2 mM Ca2+ (H/BIS + 2Ca). The rate of exocytosis was measured for 3 min in controls and 5 min for inhibitors or inhibitors with Ca2+. Cells were loaded with dopamine.

Mentions: Both kinase inhibitors depressed relative exocytosis in control experiments. It was reduced to 0.64 ± 0.05 by H-89 (n = 27) and to 0.76 ± 0.06 (n = 24) by BIS (Fig. 9 A). The depression implies either that there is some background activation of the kinases in unstimulated cells or that the inhibitors have nonspecific actions at other steps in exocytosis. Therefore, in the statistical tests below, we use the H-89- or BIS-inhibited measurement as the control value for calculating the effect of subsequently added Ca2+.


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)

Possible role of protein kinases in Ca2+-induced exocytosis. Bars represent relative exocytosis compared with either untreated control recording (closed bars) or recording obtained with the inhibitors H-89 or BIS (open bars). All statistical tests compare the data in the open bars with calcium treatments in uninhibited cells. (A) Exocytosis induced by small [Ca2+]i elevations. Relative rates of exocytosis induced by 300 μM Ca2+ alone (0.3Ca), or after incubation for 5 min with 10 μM H-89 (H-89), H-89 with 300 μM Ca2+ (H-89 + 0.3Ca), 500 nM BIS (BIS), or BIS with 300 μM Ca2+ (BIS + 0.3Ca). (B) Exocytosis induced by higher [Ca2+]i. Relative rates of exocytosis with 2 mM Ca2+ alone (2Ca), H-89 with 2 mM Ca2+ (H-89 + 2Ca), BIS with 2 mM Ca2+ (BIS + 2Ca), combined treatments of H-89 and BIS (H/BIS), or H-89 and BIS with 2 mM Ca2+ (H/BIS + 2Ca). The rate of exocytosis was measured for 3 min in controls and 5 min for inhibitors or inhibitors with Ca2+. Cells were loaded with dopamine.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Possible role of protein kinases in Ca2+-induced exocytosis. Bars represent relative exocytosis compared with either untreated control recording (closed bars) or recording obtained with the inhibitors H-89 or BIS (open bars). All statistical tests compare the data in the open bars with calcium treatments in uninhibited cells. (A) Exocytosis induced by small [Ca2+]i elevations. Relative rates of exocytosis induced by 300 μM Ca2+ alone (0.3Ca), or after incubation for 5 min with 10 μM H-89 (H-89), H-89 with 300 μM Ca2+ (H-89 + 0.3Ca), 500 nM BIS (BIS), or BIS with 300 μM Ca2+ (BIS + 0.3Ca). (B) Exocytosis induced by higher [Ca2+]i. Relative rates of exocytosis with 2 mM Ca2+ alone (2Ca), H-89 with 2 mM Ca2+ (H-89 + 2Ca), BIS with 2 mM Ca2+ (BIS + 2Ca), combined treatments of H-89 and BIS (H/BIS), or H-89 and BIS with 2 mM Ca2+ (H/BIS + 2Ca). The rate of exocytosis was measured for 3 min in controls and 5 min for inhibitors or inhibitors with Ca2+. Cells were loaded with dopamine.
Mentions: Both kinase inhibitors depressed relative exocytosis in control experiments. It was reduced to 0.64 ± 0.05 by H-89 (n = 27) and to 0.76 ± 0.06 (n = 24) by BIS (Fig. 9 A). The depression implies either that there is some background activation of the kinases in unstimulated cells or that the inhibitors have nonspecific actions at other steps in exocytosis. Therefore, in the statistical tests below, we use the H-89- or BIS-inhibited measurement as the control value for calculating the effect of subsequently added Ca2+.

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