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Protein kinase activation increases insulin secretion by sensitizing the secretory machinery to Ca2+.

Wan QF, Dong Y, Yang H, Lou X, Ding J, Xu T - J. Gen. Physiol. (2004)

Bottom Line: Phorbol esters and forskolin also increase the size of RRP to a lesser extent.The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases.The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.

ABSTRACT
Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca(2+), we characterize three kinetically different pools of vesicles in rat pancreatic beta-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is approximately 20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca(2+), we find that the HCSP is a distinct population of vesicles from those colocalized with Ca(2+) channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca(2+).

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The rate constants and amplitudes of exponential fits to Cm responses with similar post-flash [Ca2+]i levels are averaged and plotted versus [Ca2+]i. Circles and triangles represent the fast and slow components of double exponential fits. For [Ca2+]i > 10 μM, it is hard to distinguish the HCSP from RRP, therefore we only fit a single exponential to the Cm traces and the rate constants are denoted as squares. The open and filled symbols represent data from control and PMA-treated cells, respectively. The Ca2+ dependence of the rate of exocytosis from the HCSP was fitted by the equation (dashed line) Rate = Rmax/(1 + (Kd/[Ca2+]i)n), where Rmax, Kd, and n were 69.4 ± 8.8 s−1, 2.5 ± 0.5 μM, and 1.9 ± 0.7, respectively. The dotted lines in the upper panel mark the averaged HCSP sizes for control and PMA-treated cells.
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fig3: The rate constants and amplitudes of exponential fits to Cm responses with similar post-flash [Ca2+]i levels are averaged and plotted versus [Ca2+]i. Circles and triangles represent the fast and slow components of double exponential fits. For [Ca2+]i > 10 μM, it is hard to distinguish the HCSP from RRP, therefore we only fit a single exponential to the Cm traces and the rate constants are denoted as squares. The open and filled symbols represent data from control and PMA-treated cells, respectively. The Ca2+ dependence of the rate of exocytosis from the HCSP was fitted by the equation (dashed line) Rate = Rmax/(1 + (Kd/[Ca2+]i)n), where Rmax, Kd, and n were 69.4 ± 8.8 s−1, 2.5 ± 0.5 μM, and 1.9 ± 0.7, respectively. The dotted lines in the upper panel mark the averaged HCSP sizes for control and PMA-treated cells.

Mentions: Next, we tested the effect of PMA, a PKC activator, on exocytosis in pancreatic β-cells. A prominent HCSP component with much larger size than in control cells is evident after PMA treatment (Fig. 2, A and B). When the averaged time courses of Cm increase in response to similar post-flash [Ca2+]i levels were compared, PMA clearly increased the burst component of exocytosis as well as the sustained component (Fig. 2 C). To investigate the Ca2+ dependence of secretion, the exocytotic bursts were further fitted by a double exponential to obtain the rate constants and amplitudes of the HCSP and RRP. Fig. 3 compared the sizes and the rate constants of the HCSP and RRP from control (n = 93) and PMA-treated (n = 117) cells between [Ca2+]i levels of 0.7 and 30 μM. Despite the comparable rate constants, the amplitudes of the HCSP were much greater in PMA-treated cells, as shown in the upper part of Fig. 3. We did not observe a clear dependence of the amplitude on [Ca2+]i.


Protein kinase activation increases insulin secretion by sensitizing the secretory machinery to Ca2+.

Wan QF, Dong Y, Yang H, Lou X, Ding J, Xu T - J. Gen. Physiol. (2004)

The rate constants and amplitudes of exponential fits to Cm responses with similar post-flash [Ca2+]i levels are averaged and plotted versus [Ca2+]i. Circles and triangles represent the fast and slow components of double exponential fits. For [Ca2+]i > 10 μM, it is hard to distinguish the HCSP from RRP, therefore we only fit a single exponential to the Cm traces and the rate constants are denoted as squares. The open and filled symbols represent data from control and PMA-treated cells, respectively. The Ca2+ dependence of the rate of exocytosis from the HCSP was fitted by the equation (dashed line) Rate = Rmax/(1 + (Kd/[Ca2+]i)n), where Rmax, Kd, and n were 69.4 ± 8.8 s−1, 2.5 ± 0.5 μM, and 1.9 ± 0.7, respectively. The dotted lines in the upper panel mark the averaged HCSP sizes for control and PMA-treated cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234026&req=5

fig3: The rate constants and amplitudes of exponential fits to Cm responses with similar post-flash [Ca2+]i levels are averaged and plotted versus [Ca2+]i. Circles and triangles represent the fast and slow components of double exponential fits. For [Ca2+]i > 10 μM, it is hard to distinguish the HCSP from RRP, therefore we only fit a single exponential to the Cm traces and the rate constants are denoted as squares. The open and filled symbols represent data from control and PMA-treated cells, respectively. The Ca2+ dependence of the rate of exocytosis from the HCSP was fitted by the equation (dashed line) Rate = Rmax/(1 + (Kd/[Ca2+]i)n), where Rmax, Kd, and n were 69.4 ± 8.8 s−1, 2.5 ± 0.5 μM, and 1.9 ± 0.7, respectively. The dotted lines in the upper panel mark the averaged HCSP sizes for control and PMA-treated cells.
Mentions: Next, we tested the effect of PMA, a PKC activator, on exocytosis in pancreatic β-cells. A prominent HCSP component with much larger size than in control cells is evident after PMA treatment (Fig. 2, A and B). When the averaged time courses of Cm increase in response to similar post-flash [Ca2+]i levels were compared, PMA clearly increased the burst component of exocytosis as well as the sustained component (Fig. 2 C). To investigate the Ca2+ dependence of secretion, the exocytotic bursts were further fitted by a double exponential to obtain the rate constants and amplitudes of the HCSP and RRP. Fig. 3 compared the sizes and the rate constants of the HCSP and RRP from control (n = 93) and PMA-treated (n = 117) cells between [Ca2+]i levels of 0.7 and 30 μM. Despite the comparable rate constants, the amplitudes of the HCSP were much greater in PMA-treated cells, as shown in the upper part of Fig. 3. We did not observe a clear dependence of the amplitude on [Ca2+]i.

Bottom Line: Phorbol esters and forskolin also increase the size of RRP to a lesser extent.The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases.The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.

ABSTRACT
Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca(2+), we characterize three kinetically different pools of vesicles in rat pancreatic beta-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is approximately 20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca(2+), we find that the HCSP is a distinct population of vesicles from those colocalized with Ca(2+) channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca(2+).

Show MeSH
Related in: MedlinePlus