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Molecular Endocrinology, doi:10.1210/me.2004-0044
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Molecular Endocrinology 18 (7): 1658-1669
Copyright © 2004 by The Endocrine Society

Growth Hormone Promotes Ca2+-Induced Ca2+ Release in Insulin-Secreting Cells by Ryanodine Receptor Tyrosine Phosphorylation

Qimin Zhang, Martin Köhler, Shao-Nian Yang, Fan Zhang, Olof Larsson and Per-Olof Berggren

The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, S-171 76 Stockholm, Sweden

Address all correspondence and requests for reprints to: Qimin Zhang, Karolinska Institutet, Research Center, Stockholm South Hospital, SE-11883 Stockholm, Sweden. E-mail: Qimin.Zhang{at}sos.ki.se.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Elevation in cytoplasmic free Ca2+ concentration ([Ca2+]i) is a common mechanism in signaling events. An increased [Ca2+]i induced by GH, has been observed in relation to different cellular events. Little is known about the mechanism underlying the GH effect on Ca2+ handling. We have studied the molecular mechanisms underlying GH-induced rise in [Ca2+]i in BRIN-BD11 insulin-secreting cells. GH (500 ng/ml, 22 nM) induced a sustained increase in [Ca2+]i. The effect of GH on [Ca2+]i was prevented in the absence of extracellular Ca2+ and was inhibited by the ATP-sensitive K+-channel opener diazoxide and the voltage-dependent Ca2+-channel inhibitor nifedipine. However, GH failed to induce any changes in Ca2+ current and membrane potential, evaluated by patch-clamp recordings and by using voltage-sensitive dyes. When the intracellular Ca2+ pools had been depleted using the Ca2+-ATPase inhibitor thapsigargin, the effect of GH was inhibited. In addition, GH-stimulated rise in [Ca2+]i was completely abolished by ruthenium red, an inhibitor of mitochondrial Ca2+ transport, and caffeine. GH induced tyrosine phosphorylation of ryanodine receptors. The effect of GH on [Ca2+]i was completely blocked by the tyrosine kinase inhibitors genistein and lavendustin A. Interestingly, treatment of the cells with GH significantly enhanced K+-induced rise in [Ca2+]i. Hence, GH-stimulated rise in [Ca2+]i is dependent on extracellular Ca2+ and is mediated by Ca2+-induced Ca2+ release. This process is mediated by tyrosine phosphorylation of ryanodine receptors and may play a crucial role in physiological Ca2+ handling in insulin-secreting cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IT IS GENERALLY ACCEPTED that the depolarization-induced rise in cytoplasmic free Ca2+ concentration ([Ca2+]i) in the pancreatic ß-cell is mainly due to Ca2+ entry through the voltage-gated L-type Ca2+-channel. Coupling of Ca2+ entry with Ca2+ release from intracellular Ca2+ stores involves Ca2+-channels located in the endoplasmic reticulum (ER) membrane, such as inositol 1,4,5-trisphosphate receptor (IP3R)- and ryanodine receptor (RYR)-operated channels, and amplifies the Ca2+ signal induced by Ca2+ entry (1, 2). Mobilization of Ca2+ from the ER stores could be achieved by Ca2+-mediated activation of phosphatidylinositol-specific phospholipase C and thereby generation of inositol 1,4,5-trisphosphate (IP3) (3, 4) or Ca2+-induced Ca2+ release (CICR) (5, 6, 7). The latter process has been described in both muscle cells and pancreatic ß-cells (2, 4, 7).

GH is a multifunctional hormone, involved in both mitogenic and metabolic actions. GH stimulates proliferation of pancreatic ß-cells (8, 9) and enhances insulin secretion after long-term incubation (10). The initial event in GH signaling is activation of the cytoplasmic Janus tyrosine kinase JAK-2. Tyrosine phosphorylation of several proteins, including the signal transducer and activator of transcription proteins, is involved in GH actions (11, 12). An increased [Ca2+]i has been observed in different cellular events, induced by GH (13, 14, 15, 16, 17), such as carbohydrate metabolism (15), gene transcription (14), and cell growth (8, 9). In the insulin-secreting cell line INS-1, GH-induced rise in [Ca2+]i was shown to be associated with tyrosine phosphorylation of Janus kinase 2 (17). The molecular mechanisms underlying GH-regulated Ca2+ handling and the linkage between tyrosine kinase activation and increase in [Ca2+]i are not known. Signal transduction in the insulin-secreting pancreatic ß-cells is regulated by a sophisticated interplay between nutrient secretogogs and receptor-operated pathways, similar to that activated by GH. In the present study, we have therefore investigated the effect of GH on Ca2+ handling in the insulin-secreting cell line BRIN-BD11.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GH-induced rise in [Ca2+]i in insulin-secreting cells has been observed at GH concentrations from 100-1000 ng/ml (8, 17). In the present study GH, at a concentration of 500 ng/ml, induced a sustained increase in [Ca2+]i in BRIN-BD11 cells under perifusion conditions. The elevated [Ca2+]i returned to basal levels after withdrawal of the hormone. A second application of GH resulted in a similar response (Fig. 1AGo). When cells were perifused with a Ca2+-free medium in the presence of the Ca2+ chelator EGTA, the stimulatory effect of GH was abolished, whereas the cells responded to extracellular addition of ATP, which is known to increase [Ca2+]i through promotion of IP3-induced Ca2+ mobilization from intracellular Ca2+ pools (Fig. 1BGo). The dependency of the GH action on extracellular Ca2+ was further evaluated using buffers with various concentrations of Ca2+ (Fig. 1Go, C–E). The increase in [Ca2+]i was dependent on the Ca2+ concentrations in the perifusion buffer. The GH effect was almost undetectable at 100 µM Ca2+, whereas K+ still exerted a pronounced effect on [Ca2+]i (Fig. 1EGo).



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  		Fig. 1. GH-Induced Ca2+-Dependent Rise in [Ca2+]i

BRIN-BD11 cells were perifused with buffer A containing 3 mM glucose in the presence of 1.28 mM Ca2+. Addition of GH (500 ng/ml) is indicated. The cells were depolarized by 25 mM K+ at the end of the experiments. A, GH-induced rise in [Ca2+]i. B, Cells were perifused with Ca2+-free buffer in the presence of 2 mM EGTA. The Ca2+-free buffer was introduced 2 min before GH addition and was continued for 2 min after withdrawal of GH. Addition of 100 µM ATP is indicated. The effect of GH on [Ca2+]i at various concentrations of extracellular Ca2+ is shown in panels C, D, and E. Ca-0 indicates no addition of Ca2+ in the perifusion buffer. Ca-600, Ca-300, Ca-200, and Ca-100 represent the presence of extracellular Ca2+ at concentrations of 600, 300, 200, and 100 µM, respectively. Cells were perifused with buffer A in the presence of 1.28 mM Ca2+ during the time when Ca2+ concentration is not indicated in the figures. Results are expressed as 340 nm/380 nm fluorescence ratios. Representative experiments of five (A, B, and C) or four (D and E) are shown. Results were corrected and normalized as described in Materials and Methods. Variation of basal ratios was observed in different cells and measurements.

 
The effect of GH on [Ca2+]i was evaluated when the plasma membrane was hyperpolarized by diazoxide (Fig. 2Go). Under these conditions, GH failed to induce any change in [Ca2+]i, whereas [Ca2+]i was still enhanced by ATP.



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  		Fig. 2. Effect of Diazoxide on GH-Induced Rise in [Ca2+]i

Experiments were performed as described in Fig. 1Go. Diazoxide (100 µM) was added 2 min before addition of GH (500 ng/ml) and was present during GH and ATP (100 µM) stimulation. A representative experiment of six experiments is shown.

 
The involvement of voltage-gated L-type Ca2+-channels in GH-induced rise in [Ca2+]i was examined in the presence of the specific L-type Ca2+-channel inhibitor nifedipine (10 µM). Because it is well known that the L-type Ca2+-channel blocker is voltage and use dependent, the inhibitory effect of nifedipine on the L-type Ca2+-channel activity was first evaluated in cells stimulated by 25 mM K+, and it was shown that there was an efficient inhibition of the channel by the inhibitor (Fig. 3AGo). The small increase in [Ca2+]i, observed during the second and third additions of K+ in the presence of nifedipine, implies an activation of a small pool of non-L-type Ca2+-channels as both L-type and non-L-type Ca2+-channels are present in rat pancreatic ß-cells (18, 19). The effect of GH on [Ca2+]i was investigated under conditions in which the L-type Ca2+-channel was properly inhibited by nifedipine. As shown in Fig. 3BGo, the stimulatory effect of GH was completely abolished by the inhibitor, thus pointing to activation of L-type, rather than non-L-type, Ca2+-channels in the GH action. To examine a possible modulation of GH on voltage-gated Ca2+ currents, GH was applied to cells subjected to step depolarizing voltage pulses from a holding potential of –70 mV to 10 mV at 0.05 Hz to evoke maximum peak Ca2+ currents. As shown in Fig. 4AGo, whole-cell Ca2+ currents obtained during exposure to GH run down slightly, but did not significantly differ from those registered before the exposure. However, the possibility for GH to modulate submaximal Ca2+ currents could not be excluded. Therefore, the effect of GH on the current-voltage relationship of depolarization-activated Ca2+ currents was examined. Whole-cell Ca2+ currents, generated by a set of depolarizing voltage pulses (100 msec) between –60 and 50 mV in 10-mV increments from a holding potential of –70 mV, were similar in the absence or presence of GH (Fig. 4BGo). Compiled data illustrate that there was no significant difference in the absence or presence of GH in the current-voltage relationship of depolarization-activated Ca2+ currents (Fig. 4CGo).



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  		Fig. 3. Effect of the L-type Ca2+-Channel Inhibitor Nifedipine on GH-Stimulated Rise in [Ca2+]i

Experiments were performed as described in Fig. 1Go. Cells were perifused with buffer A containing 3 mM glucose and 1.28 mM Ca2+. A, The inhibitory effect of nifedipine on Ca2+-channel activity was evaluated under conditions in which [Ca2+]i was increased by stimulation with high concentrations of K+. B, A pulse of K+ (25 mM) was applied 2 min after perifusion with nifedipine (10 µM). GH was added as indicated. Similar experiments were performed in the absence of inhibitor as a control for B (panel C). Representatives of four (A) or five (B and C) experiments are shown.

 


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  		Fig. 4. Effects of GH on Voltage-Gated Ca2+ Currents in BRIN-BD11 Cells

A, The time course of voltage-gated Ca2+ currents, evoked by repetitive depolarizing voltage steps from –70 mV to 10 mV (100 msec, 0.05 Hz), before and during exposure to GH, shows no significant influence of GH on maximum peak Ca2+ currents (n = 6). Insets show individual Ca2+ current traces registered before and during exposure to GH. B, Sample whole-cell Ca2+ current traces, generated by a set of depolarizing voltage pulses (100 msec, 0.5 Hz) between –60 and 50 mV in 10 mV increments from a holding potential of –70 mV, obtained from a cell before exposure to GH (upper panel) and during exposure (lower panel). C, Summary graph of current density-voltage relationships showing that cells (n = 6) display similar Ca2+ currents before (solid circles) and during (open circles) exposure to GH. Data are presented as means ± SEM. Statistical significance was evaluated by paired Student’s t test.

 
The effect of GH on membrane potential of the cells was examined both by patch-clamp technique and by using voltage-sensitive dyes. Figure 5AGo shows a typical membrane potential recording using the patch-clamp technique. The cell displayed no significant change in membrane potential during application of GH. Similarly, measurements of membrane potential using the voltage-sensitive dye 1-(3-sulfonatopropyl)-8-[ß-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS), which allows experiments to be performed in intact cells at 37 C, did not show any effect of GH (Fig. 5BGo).



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  		Fig. 5. Effect of GH on Membrane Potential

A, Typical membrane potential recording of six obtained from a cell using the whole-cell patch-clamp technique before and during GH exposure. Tolbutamide (100 µM) was applied as a control. B, Changes in membrane potential were measured using the voltage-sensitive dye di-8-ANEPPS. Additions of GH (500 ng/ml) and K+ (25 mM) are indicated. A representative experiment of eight is shown. Due to light artifacts, short segments of the trace were excluded when changing perifusion buffers.

 
The involvement of intracellular Ca2+ pools in the GH-induced rise in [Ca2+]i was investigated after preincubation of the cells with thapsigargin to inhibit the Ca2+-ATPase in the ER, thereby depleting Ca2+ from the intracellular stores. Under these conditions, addition of carbachol, which increases [Ca2+]i by mobilizing Ca2+ from intracellular pools through activation of phospholipase C and thereby generation of IP3, failed to induce a change in [Ca2+]i. Interestingly, the GH-induced rise in [Ca2+]i was inhibited (Fig. 6Go, A and B). To further demonstrate the essential role of the intracellular Ca2+ stores in GH action, ruthenium red, a polycationic dye that is known to inhibit mitochondrial Ca2+ transport or Ca2+-release channels of the ER (20), was introduced. In the presence of ruthenium red, the GH-induced rise in [Ca2+]i was completely abolished (Fig. 6CGo). Because ruthenium red has been shown to inhibit the voltage-gated L-type Ca2+-channel (21), we investigated the effect of ruthenium red on the K+-induced rise in [Ca2+]i. At the same concentration that abolished the GH effect, ruthenium red only slightly inhibited the effect of K+ (Fig. 6DGo). In addition, the involvement of the intracellular Ca2+-releasing channels, particularly those sensitive to caffeine, was evaluated in GH action. In this study, a relatively low dose of caffeine was applied to avoid a possible effect of the compound on depolarization of the plasma membrane (22). The effect of GH on [Ca2+]i was completely eliminated by pretreatment of the cells with caffeine (Fig. 6EGo). In contrast, caffeine did not interfere with the depolarization-induced rise in [Ca2+]i (Fig. 6FGo).



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  		Fig. 6. Involvement of Intracellular Ca2+ Pools in GH Action

A, Cells were preincubated with thapsigargin (250 nM) for 30 min to deplete Ca2+ from the intracellular pools during loading of the cells with Fura-2. Measurements of [Ca2+]i were performed immediately after incubation as described in Fig. 1Go. Addition of GH (500 ng/ml) is indicated. The filling conditions of the intracellular Ca2+ pools were evaluated by addition of carbachol (Cch, 10 µM). As a control, similar experiments were performed in cells in which no thapsigargin was present during the incubation (panel B). The effect of ruthenium red on GH-induced rise in [Ca2+]i is shown in panel C. Ruthenium red (RR, 10 µM) was introduced 2 min before the second addition of GH (500 ng/ml) and was present during GH stimulation. After withdrawal of RR, the cells were subjected to additional GH stimulation to verify the response of the cells to GH. Representative experiments of five are shown. The effect of RR (10 µM) on K+-induced rise in [Ca2+]i is shown in panel D. Representative experiments of four are shown. In panels E and F, similar experiments were performed as in panel C. Caffeine (2 mM) was introduced 4 min before the second addition of GH (in panel E) or K+ (25 mM, in panel F) and was present during the GH or K+ stimulation. Representative experiments of six are shown.

 
Because the above results suggested a role of RYRs in the GH-induced increase in [Ca2+]i and the fact that tyrosine phosphorylation is a crucial event in GH action, we investigated whether GH stimulation could result in tyrosine phosphorylation of RYRs. For this purpose, RYR antibodies, which react with all three isoforms of RYRs (RYR I, II, and III), were applied. In this study, cells were incubated with GH for 2 min to obtain marked tyrosine phosphorylation of the receptor (23). GH caused a dramatic tyrosine phosphorylation of RYRs (Fig. 7AGo). Similar to that reported in T cells (23), tyrosine phosphorylation of RYRs in insulin-secreting cells was transient, being almost undetectable when cells were stimulated with GH for 10 min (data not shown). To test whether CICR could be sensitized by GH treatment, we examined the effect of prior exposure to GH on K+-induced rise in [Ca2+]i. In the experiments, cells were perifused with a buffer containing low concentration of Ca2+ (10 µM), at which high K+, but not GH, was able to raise [Ca2+]i. Under these conditions, the K+-stimulated rise in [Ca2+]i was significantly enhanced in the presence of GH (Fig. 7Go, B and C).



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  		Fig. 7. GH-Stimulated Tyrosine Phosphorylation of RYRs

Cells were stimulated with GH (500 ng/ml) for 2 min in buffer A, containing 3 mM glucose. Cells were homogenized and subjected to immunoprecipitation using polyclonal anti-RYR. The precipitate was applied to Western blotting using antiphosphotyrosine. A, GH-stimulated tyrosine phosphorylation of RYRs in the insulin-secreting cells. The mass of the protein corresponds to the mass of RYR in rat brain extracts immunoblotted by the anti-RYR. The bar plot shows the density of the phosphorylated RYR bands in the presence (solid bars) or absence (open bars) of GH. Result derived from three separate experiments (a–c) is shown. B, K+ (25 mM)-induced rise in [Ca2+]i during perifusion with 10 µM Ca2+. Representative traces of seven are shown. GH (500 ng/ml) was present 2 min before and during the second K+ stimulation as indicated. Cells were stimulated by K+ twice in both control and experimental groups, and the areas of peak 1 (P1) and peak 2 (P2) were calculated. C, Ratios of P2/P1, derived from seven separate experiments under each condition (***, P < 0.001).

 
The role of tyrosine kinase activity in the GH-induced increase in [Ca2+]i was verified in the presence of the protein tyrosine kinase inhibitors genistein (50 µM) (Fig. 8AGo) or lavendustin A (1 µM) (Fig. 8BGo). The cells were exposed to GH twice, and the effect of the protein tyrosine kinase inhibitors was evaluated during the second addition of GH. The stimulatory effect of GH on [Ca2+]i was completely abolished by the tyrosine kinase inhibitors. In contrast, genistein showed only a minor inhibition of K+-induced rise in [Ca2+]i (Fig. 8Go, C and D). This inhibition by genistein was no longer present in cells pretreated with thapsigargin (Fig. 8Go, E and F), suggesting that the effect of the tyrosine kinase inhibitor on [Ca2+]i increase reflects intracellular Ca2+ mobilization mediated by CICR.



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  		Fig. 8. Inhibitory Effect of the Tyrosine Kinase Inhibitors Genistein and Lavendustin A on GH-Stimulated Rise in [Ca2+]i

Experiments were performed as described in Fig. 1Go. Genistein (50 µM) (A) and lavendustin A (1 µM) (B) were introduced 4 min before second addition of GH and were present during stimulation of the cells with GH. Representative experiments of five (A) and four (B) are shown. K+-induced rise in [Ca2+]i and the effect of genistein (50 µM) on the effect of K+ are shown in panels C and D, respectively. Genistein was introduced in the same way as in panel A. In panels E and F, experiments similar to C and D were performed. Cells were pretreated with thapsigargin (250 nM, 30 min), and Ca2+ measurement was carried out in the absence (E) or presence (F) of genistein (50 µM) as indicated. Representative experiments of four are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Mobilization of Ca2+ from intracellular stores plays a significant role in the regulation of multiple cellular functions. We have previously reported that GH-mediated mitogenesis in the pancreatic ß-cell is associated with a rise in [Ca2+]i (8). Here we show that GH-induced rise in [Ca2+]i in insulin-secreting cells is mediated through CICR, a process that is activated by tyrosine phosphorylation of RYRs.

The presence of extracellular Ca2+ was essential for the GH-induced rise in [Ca2+]i. GH was unable to induce a stimulatory effect on [Ca2+]i when Ca2+ was excluded from the extracellular solution. The dependency on extracellular Ca2+ suggests that a certain amount of Ca2+ entry is essential for the GH-induced event. In pancreatic ß-cells, opening of voltage-gated L-type Ca2+-channels by depolarization of the plasma membrane is the most important mechanism in raising [Ca2+]i. Indeed, GH-induced rise in [Ca2+]i was abolished by diazoxide and the L-type Ca2+-channel blocker nifedipine, indicating dependency of the effect of GH on membrane potential and a requirement for functional L-type Ca2+-channels, as previously demonstrated in INS-1 cells (17). However, we have no evidence that membrane depolarization and activation of the L-type Ca2+-channel are directly involved in the GH action, as evidenced by the results from patch-clamp studies and the studies using voltage-sensitive dyes. In addition, GH failed to induce a rise in [Ca2+]i, whereas the effect of K+ was not affected when the extracellular Ca2+ concentration was reduced to 100 µM, suggesting that the mechanism behind GH-induced rise in [Ca2+]i was different from that induced by K+, in which opening of the voltage-gated Ca2+-channels by membrane depolarization is the key step. Furthermore, the effect of GH was abolished by caffeine, which had no effect on the K+-induced rise in [Ca2+]i, indicating an involvement of intracellular Ca2+ stores, rather than a direct effect on membrane potential and L-type Ca2+-channels.

Several reports have shown a crucial role of intracellular Ca2+ stores in Ca2+ handling (2, 4). Coupling Ca2+ entry to Ca2+ release from the ER may involve multiple ER Ca2+ channels. The most important intracellular Ca2+-release channels are IP3Rs and RYRs, both of which are expressed in pancreatic ß-cells (6, 24). In the present study, the inhibitory effect of thapsigargin, an ER Ca2+-ATPase inhibitor that depletes the intracellular Ca2+ pools on GH-induced rise in [Ca2+]i, indicates a crucial role of the intracellular Ca2+ pools in the GH action. Although ruthenium red was shown to slightly inhibit the L-type Ca2+-channel, the complete inhibition of the GH-induced rise in [Ca2+]i by ruthenium red suggests that the major effect of this compound is accounted for by inhibition of CICR. Furthermore, the effect of GH was completely abolished by caffeine, an agent that mobilizes Ca2+ from caffeine-sensitive pools through activation of the RYR (25, 26, 27) and that is commonly used to prove the existence of CICR in cells. GH had no longer a stimulatory effect on [Ca2+]i subsequent to the addition of caffeine (28, 29).

Both IP3R and RYR participate in mediating CICR. However, IP3R is not likely to contribute to the GH-induced rise in [Ca2+]i. Activation of IP3R requires both IP3 and Ca2+, as the receptor must bind both of the activators before the associated channel opens (30, 31). Our recent study on pancreatic ß-cells showed that stimulation of the cells with GH evokes an increased synthesis of diacylglycerol, but not IP3 production (8). The inability of GH to raise [Ca2+]i in the absence of extracellular Ca2+ does not support the involvement of phosphatidylinositol-specific phospholipase C in GH signaling (32, 33). Although a direct activation of IP3R by tyrosine phosphorylation of the receptor has been observed in T cells activated by CD3mAb (34), the inhibitory effect of caffeine indicates an involvement of caffeine- sensitive, rather than IP3-sensitive, Ca2+ pools (24) in the GH action.

Of the three isoforms of RYRs, both type 2 (RYR2) (2, 6, 35) and type 1 (RYR1) (36, 37) appear to participate in amplification of Ca2+ signaling in pancreatic ß-cells. RYR can be phosphorylated by protein kinase A (PKA) as well as Ca2+-calmodulin-dependent protein kinase (38, 39). Interestingly, the Ca2+ release channel must be phosphorylated to be in an active state (40), and phosphorylation of RYRs enhances the sensitivity of the channels to Ca2+, leading to increased channel opening (41, 42). In insulin-secreting cells, activation of glucagon-like peptide-1 receptor increases [Ca2+]i through sensitization of the ER Ca2+-release mechanism by cAMP, thereby facilitating CICR in response to Ca2+ entry through the voltage-gated L-type Ca2+-channels (27, 43). These studies suggest that serine or threonine phosphorylation, mediated by cAMP or PKA signaling pathways, could activate the CICR process. In contrast to glucagon-like peptide-1, GH does not increase cAMP levels in insulin-secreting cells, although the GH-induced rise in [Ca2+]i was inhibited by a PKA inhibitor (17). This phenomenon suggests a permissive role for PKA in the GH action. PKA is known to play an important role in modulating L-type Ca2+-channel activity (44), the major player in mediating Ca2+ entry and thereby triggering CICR in insulin-secreting cells. Recently, tyrosine phosphorylation of RYRs was also reported upon stimulation of T cell receptor /CD3 complex (23). However, whether tyrosine phosphorylation participates in the regulation of CICR is not known. The present study shows that GH stimulates tyrosine phosphorylation of RYRs, which is associated with a rise in [Ca2+]i. Although the tyrosine kinase inhibitors have been shown to exert nonspecific inhibitory effects on ion channels (45), our results suggest that such an inhibition can only account for a very minor effect on [Ca2+]i. Hence, the pronounced effects of the tyrosine kinase inhibitors on GH-induced rise in [Ca2+]i is, to a large extent, reflecting interference with Ca2+ mobilization from intracellular Ca2+ stores. This clearly suggests the involvement of tyrosine phosphorylation in CICR in insulin-secreting cells.

Apparently, activation of CICR induced by GH requires changes in both Ca2+ entry and phosphorylation of RYRs. It should be noted that Ca2+ entry through the voltage-gated Ca2+ channels is the main trigger for activation of RYRs although the gating of the Ca2+-release channel can be modulated by many factors. In addition, Ca2+ entry is important for ER Ca2+ load, which regulates the properties of RYRs. The open probability of RYRs is reduced when the luminal Ca2+ is decreased (28). The role of Ca2+ entry through the voltage-gated Ca2+-channel in CICR explains the loss of GH action on [Ca2+]i by hyperpolarization of the plasma membrane, blockage of the L-type Ca2+-channel, or in the absence or at very low concentrations of extracellular Ca2+. Tyrosine phosphorylation of RYR induced by GH made the channels available to activation by Ca2+ entering through the voltage-gated L-type Ca2+-channel. The role of tyrosine phosphorylation of RYRs in GH-induced CICR is similar to the situation when the channel is phosphorylated by PKA. cAMP-dependent phosphorylation per se does not activate the channel (40). However, PKA phosphorylation brings the RYRs to the state that is sensitive to Ca2+ entry. It has been proposed that phosphorylation of RYRs relieves the channel from Mg2+ inhibition (40) and promotes dissociation of FK506 binding protein (FKBP12.6) from RYRs, both processes significantly increasing open probability of the channel due to an increased sensitivity to Ca2+-dependent activation (41, 46). Although the molecular mechanisms by which tyrosine phosphorylation of RYRs increased open probability of the channel are not clear, our data suggest a role of tyrosine phosphorylation in switching the channel to the state that is sensitive to Ca2+.

Glucose is the major physiological stimulator of insulin-secreting cells, increasing [Ca2+]i through depolarization of the plasma membrane, followed by opening of the voltage-gated L-type Ca2+-channel. Actually, Ca2+-entry through the L-type Ca2+-channel occurs even at resting membrane potentials (47, 48). The latter allows certain amounts of Ca2+ to enter the cells already at a nonstimulatory concentration of glucose. This is in line with the observation that diazoxide promotes a lowering in basal [Ca2+]i. Even at 3 mM glucose, a large fraction of the ATP-regulated K+-channels are still closed, and addition of diazoxide will result in opening more of these channels, leading to membrane hyperpolarization. The consequence will be that the likelihood for the voltage-gated L-type Ca2+ channels to open is reduced, explaining the lowering in basal [Ca2+]i.

GH, through an interaction with its receptors, induces activation of a group of tyrosine kinases, including Janus kinase 2 and Src kinases, both of which have been implicated in regulation of ion channels (49, 50, 51). Although the type of the tyrosine kinase involved in the GH action remains to be identified, our data favor the idea that GH activates the CICR process through tyrosine phosphorylation of RYRs, thereby sensitizing the Ca2+-release channel to Ca2+ entering the cells through the voltage-gated L-type Ca2+-channel. Hence, the present study provides the first evidence that, in addition to serine/threonine protein kinases, tyrosine kinases are involved in regulation of CICR through tyrosine phosphorylation of RYRs in the insulin-secreting pancreatic ß-cell. Moreover, our data suggest that CICR, mediated through receptor-operated pathways, plays an important role in the ß-cell Ca2+ handling. Considering the similarities in signal transduction pathways activated by cytokines, the mechanism here suggested to underlie GH-induced [Ca2+]i changes may also be operative in response to signaling promoted by members of the cytokine family.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
The insulin-secreting cell line BRIN-BD11, a ß-cell-derived cell line that was established by electrofusion of RINm5F cells with New England Deaconess Hospital (NEDH) rat pancreatic islet cells and shares the major features of normal pancreatic ß-cells (52), was a generous gift from Dr. P. R. Flatt (University of Ulster, UK). Recombinant human GH was from Pharmacia & Upjohn (Stockholm, Sweden). Fura-2/acetoxymethylester, genistein, lavendustin A, ruthenium red, caffeine, EGTA, and Ponceau S solution were from Sigma Chemical Co. (St. Louis, MO). Nifedipine was from Knoll AG (Ludwigshaffen, Germany). Thapsigargin was from Calbiochem (La Jolla, CA), and diazoxide was from Schering-Plough Corp. (Kenilworth, NJ). Pluronic-127 and the voltage-sensitive dye di-8-ANEPPS were from Molecular Probes, Inc. (Eugene, OR). Rabbit polyclonal anti-RYR (H-300), protein A/G plus agarose, rat brain extracts (RYR positive control for immunoblotting), and horseradish peroxidase-labeled goat antirabbit IgG were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphosphotyrosine antibody was from Zymed Laboratories, Inc. (South San Francisco, CA), and protease inhibitor cocktail was from Roche Diagnostics GmbH (Mannheim, Germany). An enhanced luminescence kit was from Amersham Biosciences UK Ltd. (Chalfont St. Giles, Buckinghamshire, UK). Culture medium RPMI-1640 and fetal calf serum were purchased from Life Technologies Ltd. (Paisley, Scotland, UK).

Cell Culture
BRIN-BD11 cells, passage nos. ranging from 35 to 55, were maintained in RPMI-1640 tissue culture medium supplemented with 10% (vol/vol) fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

Measurement of [Ca2+]i
[Ca2+]i was measured using a microscope equipped with a photon counting photometer and connected to a SPEX fluorolog-2 system (53). BRIN-BD11 cells were placed and grown on glass coverslips for 24 h before the experiment. To load cells with Fura-2, cells on coverslips were incubated in buffer A, containing (in mM concentration) 125 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, 25 HEPES, and 0.1% BSA (pH 7.4), in the presence of 3 mM glucose and 1.5 µM Fura-2/acetoxymethylester for 30 min at 37 C. Coverslips were rinsed once in the same buffer without the Ca2+ indicator and subsequently mounted at the bottom of a perifusion chamber on the stage of an inverted epifluorescence microscope. The stage was thermostatically controlled to maintain the perifusate in the chamber at a constant temperature of 37 C. A cell cluster (three to five cells) was measured at excitation wavelengths of 340 nm (F340) and 380 nm (F380), with an emission wavelength of 510 nm during a steady perifusion (300 µl/min) of the cells with buffer A. GH (500 ng/ml, 22 nM) was introduced during perifusion of the cells with buffer A containing 3 mM glucose. Diazoxide, the L-type Ca2+-channel inhibitor nifedipine, as well as ruthenium red (10 µM), were added 2 min, and caffeine (2 mM) was added 4 min before introduction of GH or K+. Results are expressed as F340/F380 fluorescence ratios obtained every second, and corrected for by estimation of background fluorescence after quenching the fura-2 fluorescence with manganese. The results were further normalized by dividing the fluorescence ratios with those obtained when both the excitation wavelengths were set at 360 nm to compensate for variations in excitation light intensities.

Electrophysiological Recordings
Whole-cell Ca2+ currents and membrane potential were recorded by using the perforated-patch variant of the whole-cell patch-clamp technique to prevent the loss of soluble cytoplasmic components. Pipettes were pulled from borosilicate glass capillaries (Hilgenberg, Malsfeld, Germany) on a horizontal programmable puller (DMZ Universal Puller, Zeitz-Instrumente, Augsburg, Germany). Typical electrode resistance was 3–5 M{Omega}. Electrodes were filled with (in mM concentration): 76 Cs2SO4 (for Ca2+ current recordings) or 76 K2SO4 (for membrane potential recordings), 1 MgCl2, 10 KCl, 10 NaCl, and 5 HEPES (pH 7.35), as well as amphotericin B (0.24 mg/ml) to permeabilize the cell membrane and allow low-resistance electrical access without breaking the patch. To measure Ca2+ currents, the cells were bathed in a solution containing (in mM concentration): 138 NaCl, 5.6 KCl, 1.2 MgCl2, 10 CaCl2, 5 HEPES, and 10 tetraethylammonium chloride (pH 7.4, adjusted with NaOH). After a seal was obtained, the holding potential was set at –70 mV during the course of an experiment. One set of depolarizing voltage pulses (100 msec, 0.05 Hz), from a holding potential of –70 mV to a test potential of 10 mV, was used to evoke maximum peak Ca2+ currents. This protocol was used to examine possible effects of GH on maximum peak Ca2+ currents. Another set of depolarizing voltage pulses (100 msec) was made from a holding potential of –70 mV to several clamping potentials from –60 to 50 mV, in 10-mV increments at 0.5 Hz. This approach was employed to evaluate current-voltage relationships in cells bathed in the extracellular solution in the absence or presence of GH. For membrane potential recordings, the bath solution consisted of (in mM concentration) 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES, and 3 glucose (pH 7.2). Current and voltage were recorded with an Axopatch 200 amplifier (Axon Instruments, Foster City, CA) and filtered at 1 kHz. All recordings were made at 32–34 C. Acquisition and analysis of data were done using the software program pCLAMP (Axon Instruments).

Measurements of Membrane Potential Using Voltage-Sensitive Dye
Measurements of membrane potential were also performed using the voltage-sensitive dye di-8-ANEPPS (54). Cells grown on coverslips were washed with buffer A, containing 3 mM glucose without BSA. The loading solution was prepared by mixing 1 µl of 10% pluronic-127/dimethylsulfoxide (wt/vol) with 1 µl of di-8-ANEPPS stock solution (5 mM) in a dish and then adding 1 ml of buffer A, containing 3 mM glucose without BSA. The cells on the coverslip were incubated with the loading solution (final concentration of di-8-ANEPPS, 2.5 µM) for 15 min at 37 C. Coverslips were rinsed once in the same buffer and subsequently mounted at the bottom of a perifusion chamber on the stage of an inverted epifluorescence microscope. The stage was thermostatically controlled to maintain the perifusate in the chamber at a constant temperature of 37 C. A cell cluster (three to five cells) or a single cell was excited at the wavelength 475 nm, and the fluorescence emission was detected with bandpass filters at 600–640 nm (F620) and at 515–565 nm (F560) during a steady perifusion (300 µl/min) with buffer A, containing 3 mM glucose. GH (500 ng/ml) or K+ (25 mM) was introduced during perifusion. Results are expressed as F620/F560 fluorescence ratios obtained every 2 sec.

Detection of Tyrosine-Phosphorylated RYR
BRIN-BD11 cells were harvested and washed three times in buffer A. Cells were suspended in 2 ml of the buffer with 3 mM glucose and were placed in two Eppendorf tubes with 1 ml/tube, followed by an incubation in the presence or absence of GH (500 ng/ml) for 2 min, at 37 C. The incubation was terminated by removal of the buffer and addition of ice-cold homogenization buffer (23) containing (in mM concentration) 20 HEPES, 110 NaCl (pH 7.5), in the presence of protease inhibitors. After three washes in the homogenization buffer, cells were homogenized on ice in 100 µl of the buffer using a pellet pestle motor homogenizer. Cell debris was removed by centrifugation (1,000 x g, 5 min, 4 C), and the supernatant was subjected to ultracentrifugation (100,000 x g, 60 min, 4 C). The pellets from ultracentrifugation were solubilized in lysis buffer containing (in mM concentration) 25 HEPES, 150 NaCl, 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (wt/vol), pH 7.2 (23), in the presence of protease inhibitors. Aliquots (200 µg of protein) were incubated overnight at 4 C with 2 µg of rabbit polyclonal anti-RYR. The immunocomplex was precipitated by protein A/G plus-agarose, and equal amounts of the immunoprecipitated material were subjected to SDS-PAGE (6% separating gel, 3% stocking gel) under reducing conditions. To ensure migration of the RYR subunits into the gel, the gel run was extended for additional 60 min after the bromphenol blue band had reached the end of the gel. Proteins in the gel were subsequently transferred onto nitrocellulose sheets. After equal amounts of protein in each lane were further ensured by reversible staining using ponceau S solution, the nitrocellulose sheets were blocked with 5% dry fatty acid-free milk in Tris-buffered saline-Tween 20 (TBS-T) (in mM concentration: 20 Tris base; 137 NaCl, pH 7.6; with 0.05% Tween-20) overnight at 4 C and immunostained with rabbit antiphosphotyrosine antibody (1:2000) in TBS-T/1% BSA overnight, at 4 C. After extensive washes in TBS-T, the nitrocellulose membrane was incubated with horseradish peroxidase-labeled goat antirabbit IgG in TBS-T/1% BSA for 1 h at room temperature. The membrane was extensively washed, and the immunostained proteins were visualized by enhanced chemiluminescence.

Statistical Analysis
Statistical significance was tested with Student’s t test when appropriate.


   FOOTNOTES
 
This work was supported by a grant from David och Astrid Hageléns Stiftelse, by funds from Karolinska Institutet, by grants from Juvenile Diabetes Research Foundation International (JDRFI), the Novo Nordisk Foundation, National Institutes of Health Grant DK-58508, the Swedish Diabetes Association, the Family Persson Foundation, Bert von Kantzows Foundation, and the Swedish Medical Research Council.

Abbreviations: [Ca2+]i, Cytoplasmic free Ca2+ concentration; CICR, Ca2+-induced Ca2+ release; di-8-ANEPPS, 1-(3-sulfonatopropyl)-8-[ß-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium betaine; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; PKA, protein kinase A; RYR, ryanodine receptor; TBS-T, Tris-buffered saline-Tween 20.

Received for publication February 2, 2004. Accepted for publication March 24, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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