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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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Exocytosis induced by [Ca2+]i. (A) Amperometric record acquired in an ionomycin-pretreated cell as in Fig. 2 A. Solutions containing 300 μM (closed bar) or 2 mM Ca2+ (open bar) were applied as indicated. (B) Rate of exocytosis in the presence of solutions containing different added Ca2+ was averaged for 6-min recording time and compared with that in control (3–14 cells per data point). (C) Average time course of [Ca2+]i rise and exocytosis for several cells treated with ionomycin. Basal measurements were made in Ca2+-free external solution and then cells were treated with solutions containing 300 μM Ca2+. Intracellular [Ca2+]i was measured with the high-affinity dye, indo-1 AM (n = 5), and relative rate of exocytosis was averaged from 12 cells.
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Figure 3: Exocytosis induced by [Ca2+]i. (A) Amperometric record acquired in an ionomycin-pretreated cell as in Fig. 2 A. Solutions containing 300 μM (closed bar) or 2 mM Ca2+ (open bar) were applied as indicated. (B) Rate of exocytosis in the presence of solutions containing different added Ca2+ was averaged for 6-min recording time and compared with that in control (3–14 cells per data point). (C) Average time course of [Ca2+]i rise and exocytosis for several cells treated with ionomycin. Basal measurements were made in Ca2+-free external solution and then cells were treated with solutions containing 300 μM Ca2+. Intracellular [Ca2+]i was measured with the high-affinity dye, indo-1 AM (n = 5), and relative rate of exocytosis was averaged from 12 cells.

Mentions: We wanted to estimate the dependence of exocytosis on the mean [Ca2+]i achieved with ionomycin treatment. Initial experiments with indo-1 had shown that application of 2 mM Ca2+ in the presence of ionomycin raised [Ca2+]i into the range where the response of indo-1 saturates. Therefore, we switched to mag-indo-1-AM, a dye with much lower Ca2+ affinity. In various cells exposed to 2 mM Ca2+, [Ca2+]i increased monotonically, after a delay of ∼1 min (Fig. 2C and Fig. D). The average [Ca2+]i reported by this dye at the end of the 3-min Ca2+ stimulation was 313 ± 74 μM (n = 8). Although [Ca2+]i rose for several minutes, exocytosis developed early, before a large change in the mag-indo-1 signal was observed, indicating that exocytosis can be efficiently induced at low micromolar concentrations of Ca2+ (Fig. 2 D). To improve our estimate, we examined exocytosis in ionomycin with several lower Ca2+ concentrations in the bathing solution (Fig. 3A and Fig. B). The 1 mM Ca2+ was about as effective as 2 mM, and 300 μM elicited a half-maximal rate of exocytosis. After these 3-min incubations in 5 μM ionomycin, the [Ca2+]i was definitely not proportional to extracellular [Ca2+]. With 1 mM Ca2+ outside, the [Ca2+]i rose to 107 ± 25 μM (n = 9), whereas with 300 and 100 μM outside, it reached only 419 ± 41 nM (n = 5) and 176 ± 13 nM (n = 6), respectively. (The first value was measured with mag-indo-1 and the latter two with indo-1.) Apparently, cellular Ca2+ clearance mechanisms remained relatively effective with the lower bath [Ca2+], and they seemed to become overwhelmed if [Ca2+]i rose above 5 μM for many seconds. This result led us to use higher concentrations of ionomycin, longer incubations, and Ca2+ clearance inhibitors when calibrating the dyes (see methods). In any case, exocytosis from the pancreatic epithelial cells is quite sensitive to Ca2+ since it reaches significant levels already with [Ca2+]i as low as 300–400 nM (Fig. 3 C).


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)

Exocytosis induced by [Ca2+]i. (A) Amperometric record acquired in an ionomycin-pretreated cell as in Fig. 2 A. Solutions containing 300 μM (closed bar) or 2 mM Ca2+ (open bar) were applied as indicated. (B) Rate of exocytosis in the presence of solutions containing different added Ca2+ was averaged for 6-min recording time and compared with that in control (3–14 cells per data point). (C) Average time course of [Ca2+]i rise and exocytosis for several cells treated with ionomycin. Basal measurements were made in Ca2+-free external solution and then cells were treated with solutions containing 300 μM Ca2+. Intracellular [Ca2+]i was measured with the high-affinity dye, indo-1 AM (n = 5), and relative rate of exocytosis was averaged from 12 cells.
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Related In: Results  -  Collection

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Figure 3: Exocytosis induced by [Ca2+]i. (A) Amperometric record acquired in an ionomycin-pretreated cell as in Fig. 2 A. Solutions containing 300 μM (closed bar) or 2 mM Ca2+ (open bar) were applied as indicated. (B) Rate of exocytosis in the presence of solutions containing different added Ca2+ was averaged for 6-min recording time and compared with that in control (3–14 cells per data point). (C) Average time course of [Ca2+]i rise and exocytosis for several cells treated with ionomycin. Basal measurements were made in Ca2+-free external solution and then cells were treated with solutions containing 300 μM Ca2+. Intracellular [Ca2+]i was measured with the high-affinity dye, indo-1 AM (n = 5), and relative rate of exocytosis was averaged from 12 cells.
Mentions: We wanted to estimate the dependence of exocytosis on the mean [Ca2+]i achieved with ionomycin treatment. Initial experiments with indo-1 had shown that application of 2 mM Ca2+ in the presence of ionomycin raised [Ca2+]i into the range where the response of indo-1 saturates. Therefore, we switched to mag-indo-1-AM, a dye with much lower Ca2+ affinity. In various cells exposed to 2 mM Ca2+, [Ca2+]i increased monotonically, after a delay of ∼1 min (Fig. 2C and Fig. D). The average [Ca2+]i reported by this dye at the end of the 3-min Ca2+ stimulation was 313 ± 74 μM (n = 8). Although [Ca2+]i rose for several minutes, exocytosis developed early, before a large change in the mag-indo-1 signal was observed, indicating that exocytosis can be efficiently induced at low micromolar concentrations of Ca2+ (Fig. 2 D). To improve our estimate, we examined exocytosis in ionomycin with several lower Ca2+ concentrations in the bathing solution (Fig. 3A and Fig. B). The 1 mM Ca2+ was about as effective as 2 mM, and 300 μM elicited a half-maximal rate of exocytosis. After these 3-min incubations in 5 μM ionomycin, the [Ca2+]i was definitely not proportional to extracellular [Ca2+]. With 1 mM Ca2+ outside, the [Ca2+]i rose to 107 ± 25 μM (n = 9), whereas with 300 and 100 μM outside, it reached only 419 ± 41 nM (n = 5) and 176 ± 13 nM (n = 6), respectively. (The first value was measured with mag-indo-1 and the latter two with indo-1.) Apparently, cellular Ca2+ clearance mechanisms remained relatively effective with the lower bath [Ca2+], and they seemed to become overwhelmed if [Ca2+]i rose above 5 μM for many seconds. This result led us to use higher concentrations of ionomycin, longer incubations, and Ca2+ clearance inhibitors when calibrating the dyes (see methods). In any case, exocytosis from the pancreatic epithelial cells is quite sensitive to Ca2+ since it reaches significant levels already with [Ca2+]i as low as 300–400 nM (Fig. 3 C).

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