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Muscarinic modulation of voltage-dependent Ca2+ channels in insulin-secreting HIT-T15 cells
J A Love1, N W Richards, C Owyang
1Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson 39216, USA.
Abstract:
Potentiation of insulin secretion from pancreatic beta-cells by acetylcholine requires ongoing cyclic electrical activity initiated by other depolarizing secretagogues. Patch-clamp recordings in glucose-free solutions were made from the clonal beta-cell line HIT-T15 to determine whether the muscarinic agonist bethanechol (BCh) modulated voltage-dependent Ca2+ channels independent of effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type) Ca2+ channels with a mean conductance of 26 pS were observed in cell-attached patches. BCh (100 microM) caused a two- to threefold increase in both fractional open time and mean current of single Ca2+ channels. These changes resulted from a 44% decrease in the longer of two apparent mean closed times and a 25% increase in the mean open time. Similar BCh-stimulated increases in macroscopic Ca2+ currents were recorded in whole cell, perforated-patch recordings. The role of protein kinase C (PKC) in the muscarinic activation of Ca2+ channels was tested using a variety of PKC activators and inhibitors. Acute application of either the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the membrane-permeable diacylglycerol analog 1,2-didecanoyl-rac-glycerol mimicked the effects of BCh, whereas an inactive phorbol (4 alpha) had no effect. Depletion of PKC activity by chronic exposure to PMA or acute application of the PKC inhibitor staurosporine greatly reduced or abolished muscarinic activation of Ca2+ channels. These results are consistent with muscarinic activation of L-type, voltage-dependent Ca2+ channels mediated in large part by PKC.
Insights
Acetylcholine enhances insulin secretion by modulating pancreatic beta-cell L-type calcium channels. This potentiation, mediated by protein kinase C (PKC), involves increased channel open time and current.
Area of Science:
- Endocrinology
- Cell Biology
- Neuroscience
Background:
- Insulin secretion from pancreatic beta-cells is crucial for glucose homeostasis.
- Acetylcholine potentiates insulin secretion, but the underlying ionic mechanisms are not fully understood.
- Understanding these mechanisms is vital for developing treatments for diabetes.
Purpose of the Study:
- To investigate whether the muscarinic agonist bethanechol (BCh) modulates voltage-dependent Ca2+ channels in pancreatic beta-cells independently of membrane potential changes.
- To elucidate the role of protein kinase C (PKC) in the muscarinic potentiation of Ca2+ channel activity.
Main Methods:
- Patch-clamp recordings were performed on the HIT-T15 beta-cell line in glucose-free solutions.
- Single-channel and whole-cell currents of high-threshold, dihydropyridine-sensitive (L-type) Ca2+ channels were analyzed.
- The effects of BCh, PKC activators (phorbol ester, diacylglycerol analog), and PKC inhibitors (staurosporine) were assessed.
Main Results:
- Bethanechol (BCh) significantly increased both the fractional open time and mean current of single L-type Ca2+ channels.
- BCh altered channel kinetics by decreasing closed time and increasing open time.
- PKC activators mimicked BCh's effects, while PKC inhibition abolished them, indicating a crucial role for PKC.
Conclusions:
- Muscarinic activation of L-type Ca2+ channels in pancreatic beta-cells is largely mediated by protein kinase C (PKC).
- This pathway contributes to the potentiation of insulin secretion by acetylcholine.
- The findings provide insights into the regulation of beta-cell function and insulin secretion.