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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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Stimulation of exocytosis by Ca2+ entry. (A) Amperometric record. Dopamine-loaded cells were treated with 5 μM ionomycin in Ca2+-free external saline solution containing 100 μM EGTA for >6 min before the recording. External solution containing 2 mM Ca2+ was applied as indicated by the bar. Ionomycin was present throughout the recording. The large deflection near 7 min may represent breakdown of a nearby cell releasing a large quantity of dopamine into the medium. (B) Rate of exocytosis for the recording in A. The horizontal broken line indicates average rate of exocytosis in the control period. (C) Simultaneous Ca2+ measurement from the same cell using a low-affinity Ca2+ dye, mag-indo 1-AM. (D) Averaged kinetics of Ca2+ rise and exocytosis for many cells. [Ca2+]i (n = 8, line) and relative rate of exocytosis (n = 16, •) measured as 2 mM Ca2+ was applied to cells (horizontal bar). Basal [Ca2+]i and the normalized rate of exocytosis (1.0) in the control period are indicated by a horizontal broken line. Error bars are shown only in the downward direction.
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Figure 2: Stimulation of exocytosis by Ca2+ entry. (A) Amperometric record. Dopamine-loaded cells were treated with 5 μM ionomycin in Ca2+-free external saline solution containing 100 μM EGTA for >6 min before the recording. External solution containing 2 mM Ca2+ was applied as indicated by the bar. Ionomycin was present throughout the recording. The large deflection near 7 min may represent breakdown of a nearby cell releasing a large quantity of dopamine into the medium. (B) Rate of exocytosis for the recording in A. The horizontal broken line indicates average rate of exocytosis in the control period. (C) Simultaneous Ca2+ measurement from the same cell using a low-affinity Ca2+ dye, mag-indo 1-AM. (D) Averaged kinetics of Ca2+ rise and exocytosis for many cells. [Ca2+]i (n = 8, line) and relative rate of exocytosis (n = 16, •) measured as 2 mM Ca2+ was applied to cells (horizontal bar). Basal [Ca2+]i and the normalized rate of exocytosis (1.0) in the control period are indicated by a horizontal broken line. Error bars are shown only in the downward direction.

Mentions: Vesicular release of catecholamine was monitored as pulses of electric current generated by oxidation of the molecules at the tip of a carbon-fiber electrode polarized at +600 mV. Carbon-fiber microelectrodes were fabricated from 5–11-μm carbon fibers (PAN T650 or P25; Amoco Performance Products) and polypropylene 10 μl micropipettor tips as described by Koh and Hille 1999. Application of +600 mV to a fresh electrode in the bath elicited an initial transient polarization current. Measurements were begun after this electrode current fell below 10 pA. The amperometric currents were recorded with EPC 5 or EPC 9 (HEKA Elektronik) patch-clamp amplifiers, filtered at 10 kHz (−3 dB cut off frequency) using a four-pole Bessel filter (Ithaco Co.), digitized, and stored on video tape. For analysis, the recordings were replayed, filtered, digitized, and stored by a computer. The final filter frequency was 100 Hz except where indicated. The sampling rate was four or five times faster than the filter frequency. In simultaneous recordings of [Ca2+]i and amperometry, illumination of the carbon-fiber probe with ultraviolet light produced small current artifacts that were digitally corrected when larger than the baseline noise level (see Fig. 2, Fig. 4, and Fig. 7).


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)

Stimulation of exocytosis by Ca2+ entry. (A) Amperometric record. Dopamine-loaded cells were treated with 5 μM ionomycin in Ca2+-free external saline solution containing 100 μM EGTA for >6 min before the recording. External solution containing 2 mM Ca2+ was applied as indicated by the bar. Ionomycin was present throughout the recording. The large deflection near 7 min may represent breakdown of a nearby cell releasing a large quantity of dopamine into the medium. (B) Rate of exocytosis for the recording in A. The horizontal broken line indicates average rate of exocytosis in the control period. (C) Simultaneous Ca2+ measurement from the same cell using a low-affinity Ca2+ dye, mag-indo 1-AM. (D) Averaged kinetics of Ca2+ rise and exocytosis for many cells. [Ca2+]i (n = 8, line) and relative rate of exocytosis (n = 16, •) measured as 2 mM Ca2+ was applied to cells (horizontal bar). Basal [Ca2+]i and the normalized rate of exocytosis (1.0) in the control period are indicated by a horizontal broken line. Error bars are shown only in the downward direction.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230622&req=5

Figure 2: Stimulation of exocytosis by Ca2+ entry. (A) Amperometric record. Dopamine-loaded cells were treated with 5 μM ionomycin in Ca2+-free external saline solution containing 100 μM EGTA for >6 min before the recording. External solution containing 2 mM Ca2+ was applied as indicated by the bar. Ionomycin was present throughout the recording. The large deflection near 7 min may represent breakdown of a nearby cell releasing a large quantity of dopamine into the medium. (B) Rate of exocytosis for the recording in A. The horizontal broken line indicates average rate of exocytosis in the control period. (C) Simultaneous Ca2+ measurement from the same cell using a low-affinity Ca2+ dye, mag-indo 1-AM. (D) Averaged kinetics of Ca2+ rise and exocytosis for many cells. [Ca2+]i (n = 8, line) and relative rate of exocytosis (n = 16, •) measured as 2 mM Ca2+ was applied to cells (horizontal bar). Basal [Ca2+]i and the normalized rate of exocytosis (1.0) in the control period are indicated by a horizontal broken line. Error bars are shown only in the downward direction.
Mentions: Vesicular release of catecholamine was monitored as pulses of electric current generated by oxidation of the molecules at the tip of a carbon-fiber electrode polarized at +600 mV. Carbon-fiber microelectrodes were fabricated from 5–11-μm carbon fibers (PAN T650 or P25; Amoco Performance Products) and polypropylene 10 μl micropipettor tips as described by Koh and Hille 1999. Application of +600 mV to a fresh electrode in the bath elicited an initial transient polarization current. Measurements were begun after this electrode current fell below 10 pA. The amperometric currents were recorded with EPC 5 or EPC 9 (HEKA Elektronik) patch-clamp amplifiers, filtered at 10 kHz (−3 dB cut off frequency) using a four-pole Bessel filter (Ithaco Co.), digitized, and stored on video tape. For analysis, the recordings were replayed, filtered, digitized, and stored by a computer. The final filter frequency was 100 Hz except where indicated. The sampling rate was four or five times faster than the filter frequency. In simultaneous recordings of [Ca2+]i and amperometry, illumination of the carbon-fiber probe with ultraviolet light produced small current artifacts that were digitally corrected when larger than the baseline noise level (see Fig. 2, Fig. 4, and Fig. 7).

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