HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/2/301 04-2621fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gustafsson, A. J. Articles by Islam, Md. S. Search for Related Content PubMed PubMed Citation Articles by Gustafsson, A. J. Articles by Islam, Md. S.

Ryanodine receptor-operated activation of TRP-like channels can trigger critical Ca²⁺ signaling events in pancreatic ß-cells

Amanda Jabin Gustafsson¹, Hanna Ingelman-Sundberg^*, Mensur Dzabic, Justina Awasum, Nguyen Khanh Hoa^*, Claes-Göran Östenson^*, Cristina Pierro, Patrizia Tedeschi, Orison Woolcott, Shiue Chiounan, Per-Eric Lund, Olof Larsson and Md. Shahidul Islam

Karolinska Institutet,
^* Department of Molecular Medicine, Karolinska Hospital; Department of Medicine, Stockholm Söder Hospital, Research Center, Stockholm, Sweden; and
AstraZeneca R&D, Södertälje, Sweden

¹Correspondence: Department of Medicine, Karolinska Inst., Stockholm Söder Hospital, Stockholm 118 83, Sweden. E-mail: amajab{at}ki.se

SPECIFIC AIMS

Pancreatic ß-cells possess ryanodine (RY) receptors, but there is little detailed information available concerning the link between the activation of these channels to membrane excitability and downstream Ca²⁺ signaling events. We tested the hypothesis that activation of RY receptors may lead to activation of the transient receptor potential (TRP) channels and that such activation may contribute to membrane depolarization, Ca²⁺ entry through voltage-gated Ca²⁺ channels, and Ca²⁺-induced Ca²⁺ release (CICR) in the ß-cells.

PRINCIPAL FINDINGS

1. Activation of RY receptors triggers Ca²⁺ entry through TRP-like channels
Activation of RY receptors by 9-methyl 5,7-dibromo eudistomin D (MBED) in the presence of glucose (10 mM) increased [Ca²⁺]_i in INS-1E cells in a characteristic pattern (Fig. 1 A). There was an initial rapid increase of [Ca²⁺]_i followed by a prolonged [Ca²⁺]_i plateau. A third feature of MBED-induced [Ca²⁺]_i increase was large regenerative [Ca²⁺]_i spikes superimposed on the [Ca²⁺]_i plateau. Similar [Ca²⁺]_i changes were observed when we used primary ß-cells obtained from Wistar rats (Fig. 1B ). In the absence of extracellular Ca²⁺, MBED caused a transient increase of [Ca²⁺]_i and the [Ca²⁺]_i plateau was absent (Fig. 1C ). The plateau phase of [Ca²⁺]_i increase was completely blocked by SKF 96365, which blocks several TRP channels. When applied in the presence of SKF 96365 (10 µM), MBED induced only a transient [Ca²⁺]_i increase that was abolished by thapsigargin. La³⁺ or Gd³⁺ abolished the [Ca²⁺]_i plateau that followed activation of RY receptors. Niflumic acid and 2-aminoethoxydiphenyl borate (2-APB), which inhibit several TRP channels, inhibited the [Ca²⁺]_i plateau. The [Ca²⁺]_i plateau was also inhibited when MBED was applied to cells depolarized by KCl (30 mM).

View larger version (23K): [in this window] [in a new window]   Figure 1. Activation of RY receptors caused a characteristic pattern of [Ca2+]i changes. [Ca2+]i was measured from fura-2-loaded single rat insulinoma cells. A) Activation of RY receptor by MBED resulted in a characteristic pattern of changes in [Ca2+]i. After addition of MBED (50 µM) in the presence 10 mM glucose, there was an initial rapid rise of [Ca2+]i, followed by a plateau. Superimposed on the [Ca2+]i plateau was a series of large [Ca2+]i spikes. B) Similar [Ca2+]i changes were observed when MBED was applied to primary ß-cells obtained from Wistar rats. C) MBED increased [Ca2+]i only transiently when extracellular Ca2+ was chelated. Note that the scales are different in the graphs. Traces are representative of experiments repeated at least 10 times.

Nimodipine did not inhibit the initial rapid rise of [Ca²⁺]_i and the [Ca²⁺]_i plateau that followed activation of RY receptors. Ruthenium red (10 µM), a blocker of TRPV channels, did not reduce the [Ca²⁺]_i plateau induced by MBED.

These results suggest that activation of RY receptors by MBED releases Ca²⁺ from the ER as well as activates Ca²⁺ influx across the plasma membrane. The fact that Ca²⁺ influx was inhibited by SKF 96365, La³⁺, Gd³⁺, 2-APB, and niflumic acid suggests that Ca²⁺ entry after activation of RY receptors is likely due to Ca²⁺ entry through channels that belong to the TRP family.

2. Activation of TRP-like channels that follows activation of RY receptors is not due to depletion of ER Ca²⁺ stores
We investigated whether activation of the phosphatidylinositol-specific phospholipase C (PI-PLC) and IP₃ system could be involved in MBED-induced [Ca²⁺]_i changes. Carbachol (100 µM), a muscarinic agonist that activates PI-PLC, caused a biphasic increase in [Ca²⁺]_i with an initial peak reflecting Ca²⁺ release from the ER and a small plateau phase representing the Ca²⁺ influx. Application of MBED in the presence of carbachol resulted in a pattern of [Ca²⁺]_i increase that was similar to that observed when MBED was used without prior application of carbachol. The [Ca²⁺]_i plateau induced by MBED was larger than that induced by carbachol. These results suggest that activation of RY receptors triggers [Ca²⁺]_i-influx mechanisms that are different from those triggered by agonists that engage the PI-PLC and IP₃ pathway.

To test whether activation of TRP channels was due to depletion of the ER or due to an increase of [Ca²⁺]_i, we first depleted ER Ca²⁺ pools by treatment with thapsigargin. Under such condition, MBED would activate RY receptors but such activation would not deplete the ER Ca²⁺ pool further. There would be no release of Ca²⁺ from the ER and thus no increase of [Ca²⁺]_i attributable to release of Ca²⁺. Activation of RY receptors in thapsigargin-treated cells resulted in a plateau-like increase in [Ca²⁺]_i. This [Ca²⁺]_i plateau was due to Ca²⁺ entry through the plasma membrane since it was abolished by SKF 96365. These results suggest that activation of RY receptors may lead to activation of TRP-channels by a mechanism that can operate without involving depletion of the ER Ca²⁺ pools.

3. Activation of TRP-like channels after activation of RY receptors changes membrane potential from –80 mV to –40 mV
We tested the effect of activation of RY receptors on plasma membrane potential using perforated patch current-clamp technique and found that such activation depolarized plasma membrane potential from –80 mV to –40 mM in a reversible manner (Fig. 2 ).

View larger version (25K): [in this window] [in a new window]   Figure 2. Activation of RY receptors and consequent activation of TRP-like channels depolarized membrane potential. Membrane potential was recorded at room temperature (21–23°C) using the perforated-patch whole cell approach. Activation of RY receptors by MBED (50 µM) depolarized membrane potential from –80 mV to –40 mV.

4. Activation of RY receptors and consequent activation of TRP-like channels cause membrane depolarization, activation of L-type voltage-gated Ca²⁺ channels and Ca²⁺ induced Ca²⁺ release
Nimodipine (5 µM) inhibited regenerative [Ca²⁺]_i spikes that were superimposed on the [Ca²⁺]_i plateau, indicating that Ca²⁺ entry through the L-type voltage-gated Ca²⁺ channels is essential for generation of these spikes. Ryanodine (50 µM) inhibited regenerative [Ca²⁺]_i spikes in a use-dependent manner.

Large [Ca²⁺]_i spikes that are indicative of membrane depolarization and Ca²⁺ influx through the L-type Ca²⁺ channels were observed even when MBED was added in the presence of diazoxide, an agent that hyperpolarizes membrane potential by opening K_ATP channels. These data indicate that MBED-induced membrane depolarization was not due to inhibition of K_ATP channels by the eudistomin compound and that Ca²⁺ influx through TRP channels can depolarize membrane potential even when K_ATP channels are open.

CONCLUSIONS AND SIGNIFICANCE

Whereas much is known about the role of the PI-PLC/IP₃-pathway in mediating Ca²⁺ entry in ß-cells, little information is available concerning the role of RY receptors in triggering Ca²⁺ influx across the plasma membrane. For activation of RY receptors, previous studies have used caffeine, which inhibits plasma membrane Ca²⁺ channels including voltage-gated Ca²⁺ channels and store-operated channels. Unlike caffeine, MBED, a potent caffeine-like activator of RY receptors does not inhibit plasma membrane Ca²⁺ channels, cAMP-phosphodiesterases, IP₃ receptors, or K_ATP channels. These properties make MBED a suitable probe for mechanistic studies of RY receptors. Activation of RY receptors of ß-cells by MBED caused changes in [Ca²⁺]_i that consisted of three components: 1) an initial rapid rise of [Ca²⁺]_i; 2) a prolonged plateau of [Ca²⁺]_i; and 3) a series of [Ca²⁺]_i spikes superimposed on the plateau. Initial rapid increase of [Ca²⁺]_i was due to release of Ca²⁺ from the ER.

The most important finding of this study was that activation of RY receptors resulted in a prolonged [Ca²⁺]_i increase after the initial rise of [Ca²⁺]_i. This [Ca²⁺]_i plateau was dependent on extracellular Ca²⁺. These results demonstrate that activation of RY receptors leads to the activation of Ca²⁺ permeable channels in the plasma membrane. In thapsigargin-treated cells, MBED did not increase [Ca²⁺]_i by releasing Ca²⁺ from the ER but there was still activation of Ca²⁺ entry. This finding argues against the possibility that Ca²⁺ influx was triggered by the increase in [Ca²⁺]_i itself. It is unlikely that Ca²⁺ entry was due to activation of any RY receptors located on the plasma membrane since Ca²⁺ influx was blocked by SKF 96365, which does not block RY receptors. The Ca²⁺ entry responsible for the [Ca²⁺]_i plateau was not mediated by the voltage-gated Ca²⁺ channels since it was not blocked by nimodipine. La³⁺, Gd³⁺, SKF 96365, niflumic acid, and 2-APB, which block many TRP channels, blocked the Ca²⁺ entry that followed the activation of RY receptors. Together, these pharmacological properties suggest that the Ca²⁺ channels that are activated as a consequence of activation of RY receptors belong to the TRP family of cation channels.

Another consequence of activation of RY receptors was the appearance of regenerative [Ca²⁺]_i spikes superimposed on the [Ca²⁺]_i plateau. Ca²⁺ entry through the L-type voltage-gated Ca²⁺ channels was essential for generation of these spikes. This is evident from the fact that the spikes were abolished by nimodipine. After activation of RY receptor, membrane potential was depolarized to –40 mV as a result of Ca²⁺ current through the TRP channels (Fig. 2) . Such depolarization in turn activated L-type Ca²⁺ channels. The generation of these spikes required CICR through RY receptors. This is evident from the fact that the spikes were inhibited in a use-dependent manner by ryanodine.

The RY receptor-operated Ca²⁺ influx described in this study is much larger than the small capacitative Ca²⁺ entry observed in ß-cells after application of high concentrations of carbachol. Consistent with this, the RY receptor-operated Ca²⁺ influx readily depolarized membrane potential to the threshold potential for activation of L-type Ca²⁺ channels. The molecular identity of the Ca²⁺ channel(s) activated as a result of activation of RY receptors in ß-cells is not fully known from our study. Members of the TRP family appear to be the best candidates.

Ca²⁺ influx described in the present study is different from the store-operated Ca²⁺ entry described earlier. This is evident from the fact that prior depletion of ER stores by thapsigargin or carbachol did not eliminate the Ca²⁺ plateau elicited by RY receptor activation. This suggests that activation of RY receptors likely stimulates TRP channels through a mechanism that is not essentially dependent on the filling state of the ER. Rather, it is likely that putative TRP channels are activated by their linkage to RY receptors as has been proposed in regards to other cells. It is unclear how activation of RY receptors couples to and gates TRP channels in ß-cells. This may involve protein-protein interactions and conformational coupling.

Molecules generated from glucose metabolism (e.g., fructose 1,6-diphosphate and long-chain Acyl CoA can activate RY receptors). Thus, RY receptors are potential links between nutrient metabolism and membrane excitability. We demonstrate that activation of RY receptor by pharmacological tools leads to a series of distinct signaling events that include not only release of Ca²⁺ from the ER but activation of TRP-like channels, membrane depolarization, and Ca²⁺ entry through voltage-gated Ca²⁺ channels and CICR. CICR mediated by RY receptors has been implicated in mediating amplification of exocytosis in ß-cells. Activation of RY receptors in ß-cells may be an important signaling event that is transduced into a coherent cellular response by participation of TRP-like channels and voltage-gated Ca²⁺ channels.

View larger version (36K): [in this window] [in a new window]   Figure 3. Schematic diagram of hypothesized involvement of RY receptor and TRP-like channels in Ca2+ entry and membrane depolarization in ß-cells. The diagram illustrates a sequence of events whereby activation of RY receptors (A) leads to the activation of TRP-like channels (B), an initial membrane depolarization to –40 mV (C), activation of the L-type voltage-gated Ca2+ channels (D), CICR (E), and exocytosis (F).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2621fje;

This article has been cited by other articles:

				E. Hughes, A. K. Lee, and A. Tse Dominant Role of Sarcoendoplasmic Reticulum Ca2+-ATPase Pump in Ca2+ Homeostasis and Exocytosis in Rat Pancreatic {beta}-Cells Endocrinology, March 1, 2006; 147(3): 1396 - 1407. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/2/301 04-2621fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gustafsson, A. J. Articles by Islam, Md. S. Search for Related Content PubMed PubMed Citation Articles by Gustafsson, A. J. Articles by Islam, Md. S.