Korean J Physiol Pharmacol.  2011 Dec;15(6):383-388. 10.4196/kjpp.2011.15.6.383.

Role of Regulators of G-Protein Signaling 4 in Ca2+ Signaling in Mouse Pancreatic Acinar Cells

• Park S
• Lee SI
• Shin DM

• KMID: 2285425
• DOI: http://doi.org/10.4196/kjpp.2011.15.6.383

Abstract

Regulators of G-protein signaling (RGS) proteins are regulators of Ca2+ signaling that accelerate the GTPase activity of the G-protein alpha-subunit. RGS1, RGS2, RGS4, and RGS16 are expressed in the pancreas, and RGS2 regulates G-protein coupled receptor (GPCR)-induced Ca2+ oscillations. However, the role of RGS4 in Ca2+ signaling in pancreatic acinar cells is unknown. In this study, we investigated the mechanism of GPCR-induced Ca2+ signaling in pancreatic acinar cells derived from RGS4-/- mice. RGS4-/- acinar cells showed an enhanced stimulus intensity response to a muscarinic receptor agonist in pancreatic acinar cells. Moreover, deletion of RGS4 increased the frequency of Ca2+ oscillations. RGS4-/- cells also showed increased expression of sarco/endoplasmic reticulum Ca2+ ATPase type 2. However, there were no significant alterations, such as Ca2+ signaling in treated high dose of agonist and its related amylase secretion activity, in acinar cells from RGS4-/- mice. These results indicate that RGS4 protein regulates Ca2+ signaling in mouse pancreatic acinar cells.

Keyword

RGS4; Ca2+ signaling; Pancreatic acinar cells

MeSH Terms

Acinar Cells
Amylases
Animals
Calcium-Transporting ATPases
GTP Phosphohydrolases
GTP-Binding Proteins
Mice
Pancreas
Proteins
Receptors, Muscarinic
Reticulum
RGS Proteins
Amylases
Calcium-Transporting ATPases
GTP Phosphohydrolases
GTP-Binding Proteins
Proteins
RGS Proteins
Receptors, Muscarinic

Figure

• 

  Fig. 1 Measurement of Ca2+ mobilization in wild-type and RGS4-/- pancreatic acinar cells. Briefly, pancreatic acinar cells were isolated from WT and RGS4-/- mice and treated with various concentrations of carbachol (i.e., 0.05 to 100µM). The treatment concentration was then immediately changed to the maximum concentration (i.e., 1 mM). Ca2+ mobilization was calculated using the ratio of the calculated area below the graph of the initial carbachol concentration and that of the maximum carbachol concentration (i.e., 1 mM). Intracellular Ca2+ concentrations were measured by fluorescence microscopy using fura2-AM. In RGS4-/- cells (B), a more sensitive and enhanced response was observed than in WT cells (A). Graphical representation of the data is presented in (C). Car, carbachol.

• 

  Fig. 2 Measurement of Ca2+ oscillations in WT and RGS4-/- cells. Effects on Ca2+ oscillations after treatment of pancreatic acinar cells with 10 pM cholecystokinin (CCK) (A); graphically represented in (B). Ca2+ oscillation frequency increased in RGS4-/- cells (p<0.01). Ca2+ oscillation frequency in 0.5µM carbachol-treated cells (C); graphically represented in (D).

• 

  Fig. 3 Expression of Ca2+ signaling molecules in WT and RGS4-/- cells. Western blotting was performed using anti-SERCA2b, anti-PMCA (5F10), anti-IP3R2, and anti-IP3R3 antibodies in isolated pancreatic acinar cells (A). Increases in SERCA2b mRNA (n=4, p<0.05) were confirmed by RT-PCR (B).

• 

  Fig. 4 Measurement of Ca2+ influx/efflux and amylase secretion. During the treatment with high doses of CCK (A) and carbachol (B), regulation of cytosol Ca2+ was observed. There were no significant differences between WT and RGS4-/- acinar cells. Percent average amylase secretion was slightly higher in RGS4-/- cells than in WT cells (n=4); however, the differences were not statistically significant (C).

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