Korean J Physiol Pharmacol.  2011 Feb;15(1):53-59.

Ca(2+)-induced Ca2+ Release from Internal Stores in INS-1 Rat Insulinoma Cells

• Choi KJ
• Cho DS
• Kim JY
• Kim BJ
• Lee KM
• Kim SH
• Kim DK
• Kim SH
• Park HS

Abstract

The secretion of insulin from pancreatic beta-cells is triggered by the influx of Ca2+ through voltage-dependent Ca2+ channels. The resulting elevation of intracellular calcium ([Ca2+]i) triggers additional Ca2+ release from internal stores. Less well understood are the mechanisms involved in Ca2+ mobilization from internal stores after activation of Ca2+ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic beta-cell line, INS-1 cells. To measure cytosolic and stored Ca2+, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. [Ca2+]i was repetitively increased by caffeine stimulation in normal Ca2+ buffer. However, peak [Ca2+]i was only observed after the first caffeine stimulation in Ca2+ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in [Ca2+]i were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca2+ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca2+ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca2+ release from internal stores was activated by caffeine, Ca2+, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic beta-cells.

Keyword

INS-1; Caffeine; Ryanodine; Calcium release; CICR

MeSH Terms

Animals
Caffeine
Calcium
Carbachol
Cytosol
Indoles
Insulin
Insulinoma
Rats
Ruthenium Red
Ryanodine
Ryanodine Receptor Calcium Release Channel
Caffeine
Calcium
Carbachol
Indoles
Insulin
Ruthenium Red
Ryanodine
Ryanodine Receptor Calcium Release Channel

Figure

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  Fig. 1. Caffeine stimulated calcium mobilization from internal stores in intact INS-1 cells. The representative traces show the effects of repetitive 30 mM caffeine stimulation on [Ca2+]i increases in the presence (A) and absence (B) of extracellular Ca2+. The data were obtained from 5 and 7 separate experiments, respectively. INS-1 cells were responsive to repetitive caffeine stimulation in normal extracellular Ca2+ buffer, but only responded to the first caffeine stimulation in Ca2+ free solution. (C) A 50 μM of ruthenium red markedly reduced the [Ca2+]i peak in the absence of extracellular Ca2+. Data were normalized to control values and expressed as mean %±S.E. Asterisk indicates the value is significantly different from the corresponding value of caffeine alone (p<0.05).

• 

  Fig. 2. KCl triggered Ca2+ release from internal stores in intact INS-1 cells. (A) The representative trace shows the effect of 45 mM KCl on [Ca2+]i increases in the presence and absence of extracellular Ca2+. The data were obtained from 6 separate experiments. [Ca2+]i elevation was not observed in Ca2+ free medium. (B) Effects of CPA plus caffeine, CPA alone or caffeine alone on KCl-induced [Ca2+]i peaks in the presence of extracellular Ca2+. The data were obtained from at least 5 separate experiments. Data were normalized to the initial [Ca2+]i peak and expressed as mean % ± S.E. Asterisks indicate that the values are significantly different from the corresponding value for control (p<0.05). Intracellular Ca2+ store depletion reduced depolarization-induced Ca2+ mobilization. (C) Representative trace shows the effect of ruthenium red on KCl-induced [Ca2+]i elevations. The data were obtained from 6 separate experiments. A 50 μM of ruthenium red significantly reduced depolarization-induced Ca2+ mobilization; the effect was restored after washout of the ruthenium red.

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  Fig. 3. Effects of internal calcium store depletion on caffeine-induced calcium release. (A) The representative trace shows the 30 mM caffeine-induced [Ca2+]i rise after internal store depletion by 10 μM carbamylcholine (CCh) in Ca2+ free solution. (B) The representative trace shows the 10 μM CCh-induced [Ca2+]i rise under store depleted conditions induced by pretreatment with 30 mM caffeine in the absence of extracellular Ca2+. (C) A representative trace of the caffeine effect under store depleted condition induced by pretreatment of cells with 10 μM cyclopiazonic acid (CPA) in Ca2+ free solution. All data were obtained from at least 5 separate experiments. Caffeine failed to increase [Ca2+]i after internal Ca2+ store depletion induced by pretreatment with CCh or CPA.

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  Fig. 4. Caffeine, Ca2+ or ryanodine-induced calcium release in permeabilized INS-1 cells. (A) 10 mM caffeine (☐), 10 μM Ca2+ (⋄) or 1 μM ryanodine (Δ) significantly stimulated Ca2+ release from internal stores in permeabilized INS-1 cells. Arrows indicate the starting point of each drug perfusion. (B) Summarized Ca2+ release rates (S–1) induced by caffeine, Ca2+ or ryanodine. Data were summarized from at least 5 experiments. Asterisks indicate that the values are significantly different from the corresponding value for control (p<0.05). (C) The blocking effect of 50 μM ruthenium red on 10 μM Ca2+-induced Ca2+ release in permeabilized INS-1 cells. (D) Summarized data showing the effects of ruthenium red on Ca2+-induced Ca2+ release rates in permeabilized cells. Asterisk indicates that the value is significantly different from the corresponding value of Ca2+ (p<0.05). Ca2+ release induced by elevated Ca2+ was completely blocked by ruthenium red, a RyR blocker, in permeabilized INS-1 cells.

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  Fig. 5. The expression of ryanodine receptors in INS-1 rat insulinoma cells. The fluorescence image (A) and the bright image (B) show the expression and distribution of RyRs in the intracellular compartments. The images were obtained from 4 separate experiments. Immunocytochemistry was done using primary RyR antibody as described under experimental procedures. The scale bar is 10 μm.

Reference

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