Ca2+-induced loss of Ca2+/calmodulin-dependent protein kinase II activity in pancreatic beta -cells

Peter M. Jones and Shanta J. Persaud

Cellular and Molecular Endocrinology Group, Biomedical Sciences Division, King's College London, London W8 7AH, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Elevations in intracellular Ca2+ in electrically permeabilized islets of Langerhans produced rapid insulin secretory responses from beta -cells, but the Ca2+-induced secretion was not maintained and was irrespective of the pattern of administration of elevated Ca2+. Ca2+-insensitive beta -cells responded normally to activators of protein kinase C or cAMP-dependent kinase with increased insulin secretion. The loss of secretory responsiveness to Ca2+ was paralleled by a reduction in Ca2+-induced protein phosphorylation. This was caused by a reduction in Ca2+/calmodulin-dependent protein kinase II (CaMK II) activity in the desensitized cells, as assessed by measuring the phosphorylation of a CaMK II-specific exogenous substrate, autocamtide-2. The Ca2+-induced reductions in kinase activity and protein phosphorylation were not dependent on the activation of Ca2+-dependent protein kinases and were not caused by the activation of phosphoprotein phosphatases or of Ca2+-activated proteases. The concomitant reductions in CaMK II activity and Ca2+-induced insulin secretion suggest that the activation of CaMK II is required for normal insulin secretory responses to increased intracellular Ca2+ concentrations.

islets of Langerhans; protein phosphorylation; calcium ion

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CHANGES IN INTRACELLULAR Ca2+ are important in regulating secretion from many cell types, including insulin-secreting pancreatic beta -cells. Many stimulators of insulin release, including the physiologically important nutrients, produce elevations in beta -cell cytosolic Ca2+ (11-14). Cytosolic Ca2+ concentrations can be directly manipulated in permeabilized beta -cells, and these have provided a useful experimental model in which to study regulatory mechanisms downstream of glucose entry, metabolism, and membrane depolarization. Secretion from permeabilized beta -cells is a regulated process (18-22), and elevations in cytosolic Ca2+ alone are sufficient to initiate insulin secretion (17-22, 40, 46). We have demonstrated previously that Ca2+-induced insulin secretion from perifused, electrically permeabilized islets of Langerhans is a transient event in which the beta -cell secretory mechanism rapidly becomes desensitized to stimulatory concentrations of Ca2+, despite retaining the ability to respond normally to other second messengers (19, 20). Selective reductions in secretory responsiveness to Ca2+ have also been noted in other secretory tissues permeabilized using methods that produce relatively small pores in the plasma membrane, thus enabling the cells to retain important cellular contents and mount prolonged secretory responses (e.g., Refs. 23 and 42).

Numerous Ca2+-sensitive elements are expressed within secretory cells, and many of these have been implicated in the secretory process, but it is still uncertain how elevations in Ca2+ are sufficient to stimulate insulin secretion. However, there is strong evidence that the activation of Ca2+/calmodulin-dependent protein kinases offers an important mechanism of Ca2+ sensing in beta -cells (reviewed in Refs. 2 and 10). Pancreatic beta -cells express several kinases that are activated by Ca2+ and calmodulin, including myosin light chain kinase (MLCK; see Refs. 10, 25, 33) and Ca2+/calmodulin-dependent protein kinases II (CaMK II; see Refs. 4, 5, 10, 16, 29, 31, 41, 44, 45) and III (CaMK III; see Ref. 16). Of these three kinase activities identified in beta -cells, the multifunctional CaMK II is most likely to subserve a role in beta -cell stimulus-secretion coupling, since both CaMK III and MLCK are thought to be dedicated to the regulation of single functions (37). The loss of secretory responsiveness of electrically permeabilized islets to Ca2+ is accompanied by reductions in Ca2+-dependent phosphorylation of endogenous islet substrates for CaMK II (20), suggestive of an important role for protein phosphorylation as a transducer of Ca2+-induced insulin secretion, although the mechanism(s) underlying the reduced protein phosphorylation in Ca2+-desensitized islets is unclear. In the present studies, we have addressed whether Ca2+-induced reductions in Ca2+-dependent protein phosphorylation reflect reduced Ca2+/calmodulin-dependent protein kinase activity and whether other Ca2+-dependent enzymes such as proteases or phosphoprotein phosphatases are involved in the desensitization process.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. ATP, BSA (fraction V), calmodulin, collagenase (type XI), cAMP, dimethyl sulfoxide (DMSO), E-64, EGTA, HEPES, 3-isobutyl-1-methylxanthine (IBMX), leupeptin, 4beta -phorbol myristate acetate (PMA), myosin light chains (MLC), phenylmethylsulfonyl fluoride (PMSF) and Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK) were from Sigma Chemical (Dorset, UK). 125I-labeled Na for insulin iodination and 3H2O were from Amersham International (Buckinghamshire, UK) and [gamma -32P]ATP (3,000 Ci/mmol) was from Du Pont (Hertfordshire, UK). P81 phosphocellulose paper was from Whatman International (Kent, UK). Calyculin A (CAL), cantharidin (CAN), cypermethrin (CM), okadaic acid (OA), a synthetic peptide corresponding to CaMK II-(290---309), and the specific CaMK II substrate autocamtide-2 were purchased from Calbiochem-Novabiochem (Nottingham, UK). All other chemicals were of analytical grade from BDH (Dorset, UK). Rats (150-200 g; Sprague-Dawley) were from the Biological Services Unit of King's College London.

Islet isolation and permeabilization. Islets of Langerhans were isolated from rat pancreata by collagenase digestion (18) and were electrically permeabilized by five exposures to an electric field of 3.4 kV/cm in a buffer ("permeation buffer") containing 140 mM potassium glutamate, 15 mM HEPES, 5 mM glucose, 1 mM EGTA, and 0.5 mg/ml BSA, pH 6.6, with CaCl2 added to give a Ca2+ concentration of 50 nM (22). For experiments designed to measure Ca2+-induced insulin secretion from permeabilized islets, the permeation buffer was supplemented with 5 mM ATP and 7 mM MgSO4, since Ca2+-induced insulin secretion from permeabilized beta -cells is dependent on the presence of millimolar amounts of MgATP (3, 18-22).

Insulin secretion from permeabilized islets. Groups of 50-100 permeabilized islets were transferred to Millipore Swinnex filter chambers containing 1 µm nylon filters and perifused at a flow rate of 1 ml/min with permeation buffer containing either 50 nM or 10 µM Ca2+ supplemented, in some experiments, with PMA (500 nM) or cAMP (500 µM) plus IBMX (100 µM). PMA and IBMX were dissolved in DMSO such that the final concentration of DMSO was <0.1% (vol/vol), which did not affect insulin secretion. Perifusate samples were collected at 2-min intervals, and insulin content was measured by radioimmunoassay (21). In experiments involving rapid and frequent (2-min) switching between a perifusion medium containing 50 nM Ca2+ and one containing 10 µM Ca2+, the dead space of the perifusion chamber was reduced by using an adapted hypodermic needle hub, and the perifusate containing 10 µM Ca2+ was supplemented with 3H2O (~12 × 103 counts · min-1 · ml-1). In these experiments, perifusion was at 1 ml/min, and perifusate samples were collected at 15-s intervals. The insulin and 3H contents of each sample were measured by radioimmunoassay and liquid scintillation counting, respectively. In all experiments, the perifusion temperature was maintained at a constant 37°C by performing experiments in a temperature-controlled chamber.

Ca2+/calmodulin-dependent protein kinase activity. Ca2+/calmodulin-dependent phosphorylation of endogenous islet proteins and of exogenous substrates was determined by following the transfer of 32P from [gamma -32P]ATP into the protein substrates, as described previously (34). Groups of 100-200 permeabilized islets were incubated for 30 min (37°C) in a modified permeation buffer (no ATP, 2 mM MgSO4) containing either 50 nM Ca2+ or 10 µM Ca2+ and supplemented with protease inhibitors, protein phosphatase inhibitors, or inhibitory peptides, as required. We have demonstrated previously that the Ca2+-induced desensitization of permeabilized islets to Ca2+ is not dependent on the presence of ATP (20). ATP was therefore omitted from the incubation buffer in these experiments to prevent the activation of Ca2+-dependent protein kinases and thus rule out any possible effects on substrate depletion or on kinase autophosphorylation during this incubation period. After incubation, islets were pelleted by brief centrifugation (9,000 g, 15 s), the supernatant was discarded, and islets were disrupted by sonication (3 × 15 s, 4°C) in a buffer ("assay buffer") containing 20 mM Tris · HCl, 2 mM EDTA, 0.5 mM EGTA, 50 µg/ml leupeptin, 1 mM PMSF, and 0.1% (vol/vol) 2-mercaptoethanol, pH 7.4. Islet extracts equivalent to 30 permeabilized islets were incubated (10 min, 30°C) in the presence of 50 µg/ml calmodulin, 0.5 mM CaCl2, 11.1 mM magnesium acetate, 1 mg/ml MLC, and 100 µM [gamma -32P]ATP (specific radioactivity 1.8 Ci/mmol), in a final assay volume of 30 µl. Reactions were terminated by addition of 30 µl of a buffer containing 4% (wt/vol) SDS, 10% (vol/vol) 2-mercaptoethanol, 20% (vol/vol) glycerol, and 0.2% (wt/vol) bromphenol blue in 125 mM Tris · HCl, pH 6.8, and the samples were boiled for 3 min. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis on 10 or 15% gels, and 32P incorporation was assessed by autoradiography and densitometric scanning on a EASY 5000 system (Ultra Violet Products, London, UK).

Islet CaMK II activity was determined by measuring the phosphorylation of the specific CaMK II peptide substrate autocamtide-2 (9). Permeabilized islets were incubated for various times (1-60 min, 37°C) in a modified permeation buffer (no ATP, 2 mM MgSO4) containing either 50 nM Ca2+ or 10 µM Ca2+, after which islet extracts were prepared as described above. Islet extracts equivalent to 40 islets were incubated in the absence or presence of 10 µM autocamtide-2, 50 µg/ml calmodulin, 0.5 mM CaCl2, 11.1 mM magnesium acetate, and 50 µM [gamma -32P]ATP (specific radioactivity 3.3 Ci/mmol) in a final incubation volume of 30 µl. After incubation (10 min, 30°C), the reaction was terminated by the addition of trichloroacetic acid to a final concentration of 10% (wt/vol). Samples were incubated at 4°C for 30 min after which precipitated material was pelleted by centrifugation (10,000 g, 2 min), and the supernatants were spotted onto discs of P81 phosphocellulose paper. The phosphocellulose discs were washed with shaking in 500 ml of 75 mM H3PO4 (2 × 5 min) followed by washing in distilled water (2 × 5 min). 32P-radiolabeled autocamtide-2 adhering to the phosphocellulose discs was measured by liquid scintillation counting, and samples of the [gamma -32P]ATP used in the assay were subjected to scintillation counting to permit estimation of specific radioactivity. CaMK II activity was estimated by using the specific radioactivity of the [gamma -32P]ATP to determine total phosphate transferred to the peptide substrate after correction for nonspecific binding and converting this mass transfer term into a rate by expressing it per islet and per minute of incubation.

Unless stated, data are expressed as means ± SE. Differences between treatment groups were assessed by ANOVA and/or Bonferroni's t-test for multiple comparisons, as appropriate, and were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ca2+-induced insulin secretion from permeabilized islets. Figure 1 shows the rapid but transient increases in insulin secretion from perifused electrically permeabilized islets of Langerhans on increasing the Ca2+ concentration of the perifusion medium from 50 nM to 10 µM (Fig. 1A). Figure 1B demonstrates that both cAMP and the protein kinase C (PKC) activator PMA stimulated insulin secretion from the Ca2+-insensitive permeabilized islets, demonstrating that the loss of Ca2+-induced insulin secretion was not due to a generalized reduction in the secretory capacity of the permeabilized cells. The loss of secretory responsiveness to Ca2+ was not prevented by changing the intracellular Ca2+ in a periodic manner, as shown in Fig. 2. Thus the pattern of insulin secretion produced by alternating between 50 nM Ca2+ and 10 µM Ca2+ at 2-min intervals (Fig. 2) was remarkably similar to that observed in response to maintained elevations in Ca2+ (Fig. 1) with a rapid desensitization of the secretory response to Ca2+. Permeabilized islets produced a rapid and reversible secretory response to the first pulse of 10 µM Ca2+, a rapid but less prolonged response to the second pulse of Ca2+, and no responses to subsequent Ca2+ pulses (Fig. 2). The inclusion of trace amounts of 3H2O in the perifusion buffer containing 10 µM Ca2+ confirmed that changes in the Ca2+ concentration of the perifusate were essentially immediate (Fig. 2).


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Fig. 1.   Insulin secretion from perifused permeabilized islets of Langerhans. Electrically permeabilized islets of Langerhans were perifused with permeation buffer containing 50 nM Ca2+ to establish a basal rate of insulin secretion (0-10 min), after which the Ca2+ concentration was increased to 10 µM (arrow) and maintained at this stimulatory concentration for the duration of the experiments. A: rapid but transient increase in insulin secretion (bullet ) from electrically permeabilized islets in response to 10 µM Ca2+. B: insulin secretory response to 10 µM Ca2+ (first arrow, 10 min), followed by responses to cAMP (square , 500 µM) or to 4beta -phorbol myristate acetate (PMA, down-triangle, 500 nM) in the continued presence of 10 µM Ca2+ (second arrow, 40-70 min). Points show the mean values for 3 separate perifusion chambers. Error bars have been omitted for the sake of clarity, and SE ranged from 7-16% of the mean value for all points shown.


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Fig. 2.   Insulin secretory responses to pulsatile changes in Ca2+ concentration. Insulin secretion (bullet , left axis) from perifused permeabilized islets in response to 2-min pulses of buffer containing 10 µM Ca2+ alternating with 2-min pulses of buffer containing 50 nM Ca2+ is shown. Trace amounts of 3H2O were added to the perifusion buffer containing 10 µM Ca2+, such that subsequent measurement of 3H in the perifusate samples (15-s intervals) could be used to assess the rate of change of the Ca2+ concentrations when switching between buffers containing 50 nM Ca2+ or 10 µM Ca2+ (dotted line, right axis). Points show means (n = 2). A: pattern of insulin secretion in response to pulsatile Ca2+ was not maintained after the second 2-min pulse of 10 µM Ca2+. B: expanding the time scale clearly demonstrated a rapid and reversible secretory response to the first pulse of 10 µM Ca2+ and a less prolonged response to the second pulse of Ca2+.

Ca2+-induced reductions in Ca2+/calmodulin-dependent kinase activity in permeabilized islets. Extracts prepared from permeabilized islets that had been incubated for 1 h in the presence of a substimulatory Ca2+ concentration (50 nM) contained protein kinase activity that, in the presence of Ca2+ and exogenous calmodulin, transferred 32P from [gamma -32P]ATP to endogenous islet proteins and to exogenous MLC, as shown in Fig. 3. Major endogenous substrates for Ca2+-dependent phosphorylation migrated with apparent molecular masses of 54-57 kDa, although several other phosphorylation events were also detectable. The 54/57-kDa phosphorylated substrates in islets have been detected previously (6), and proteins of similar molecular weights have been identified as autophosphorylated CaMK II by immunoblotting (29), immunoprecipitation (44), and protein purification (31). In accordance with this identification, the Ca2+-dependent phosphorylation of the 54/57-kDa proteins was inhibited by a synthetic peptide corresponding to amino acid residues 290-309 from the sequence of CaMK II [CaMK-(290---309)], which is a selective, substrate site-directed inhibitor of CaMK II (Ref. 32; Fig. 3, lane 3). Note, however, that the phosphorylation of exogenous MLC was only partially inhibited by CaMK-(290---309), suggesting that, although this peptide fully inhibits islet CaMK II activity, it only has small inhibitory effects on endogenous islet MLCK activity (Fig. 3, lane 3).


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Fig. 3.   Ca2+/calmodulin-dependent 32P incorporation in islet extracts: effects of the autoinhibitory peptide sequence CaMK-(290---309). Autoradiographs of 32P incorporation into islet proteins and exogenous myosin light chain (MLC) separated by SDS-PAGE on a 10% gel are shown. The 54/57-kDa endogenous substrates may be autophosphorylated Ca2+/calmodulin-dependent kinase II (CaMK II), whereas the identity of the 32-kDa endogenous substrate is unknown. Lane 1 shows 32P incorporation in the absence of Ca2+ and calmodulin, whereas lane 2 shows the Ca2+/calmodulin-dependent phosphorylation of endogenous proteins and exogenous MLC. The presence of the CaMK autoinhibitory peptide sequence CaMK-(290---309) (100 µM, lane 3) greatly reduced the Ca2+/calmodulin-dependent phosphorylation of endogenous substrates. Results shown are from 1 experiment from 3 similar experiments.

The Ca2+-induced inhibition of Ca2+/calmodulin-dependent protein phosphorylation in islet extracts is shown in Fig. 4. Extracts prepared from permeabilized islets incubated for 1 h in a substimulatory concentration of Ca2+ (50 nM) showed the expected Ca2+/calmodulin-dependent phosphorylation of the major islet substrate of 54 kDa (Fig. 4, lanes 1 and 2), but this was markedly reduced in extracts prepared from permeabilized islets incubated in the presence of a stimulatory concentration of Ca2+ (10 µM; Fig. 4, lanes 3 and 4). In five separate experiments of this type, the presence of Ca2+ and calmodulin enhanced the phosphorylation of the 54-kDa substrate by 419 ± 29% (mean ± SE, P < 0.001 vs. absence of Ca2+ and calmodulin) in control extracts (50 nM Ca2+ incubation) and by 131 ± 33% in Ca2+-desensitized extracts (10 µM Ca2+ incubation, P > 0.2 vs. absence of Ca2+ and calmodulin; P < 0.001 vs. control extracts).


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Fig. 4.   CaMK-(290---309) does not prevent Ca2+-induced reduction in protein kinase activity. Autoradiograph of 32P incorporation in islet proteins in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of Ca2+ and calmodulin is shown. Extracts of permeabilized islets that had been incubated for 30 min in a buffer containing a substimulatory concentration (50 nM) of Ca2+ contained protein kinase activity that was independent of Ca2+ and calmodulin (lane 1) and Ca2+/calmodulin-dependent kinase activity that enhanced 32P incorporation into a major islet substrate of 54 kDa (lane 2). This Ca2+/calmodulin-stimulated protein phosphorylation was much reduced in extracts of permeabilized islets that had been desensitized to Ca2+ by incubation in a buffer containing a stimulatory concentration (10 µM) of Ca2+ (lanes 3 and 4). The presence of CaMK-(290---309) (100 µM) during the incubation with 10 µM Ca2+ did not prevent the Ca2+-dependent reduction in Ca2+/calmodulin-dependent protein kinase activity in the islet extract (lanes 5 and 6). Proteins were separated on a 15% gel, and the autoradiograph is typical of effects seen in 2 separate experiments.

Similar results were obtained when CaMK II activity was measured by the incorporation of 32P into the specific exogenous substrate autocamtide-2. Initial experiments demonstrated that incubation of permeabilized islets for 1 h in the presence of 10 µM Ca2+ caused a marked reduction in the CaMK II activity measured in subsequent islet extracts (10 µM Ca2+, 21.1 ± 0.9% activity of controls incubated in 50 nM Ca2+, P < 0.01, n = 4). In subsequent experiments, we measured islet CaMK II activity in extracts of permeabilized islets after various times of exposure to substimulatory (50 nM) or stimulatory (10 µM) concentrations of Ca2+. Figure 5 shows that incubating the permeabilized islets in 50 nM Ca2+ for up to 30 min had no effect on the levels of CaMK II activity but that 10 µM Ca2+ caused a rapid and marked reduction in the enzyme activity in subsequent islet extracts. CaMK II activity showed a small but significant (P < 0.05) reduction within 1 min of exposure of the permeabilized islets to 10 µM Ca2+; maximum reduction in activity was achieved within 15-30 min, and the ~80% reduction in activity at these times is in accordance with the initial data obtained after a 1-h incubation in the presence of 10 µM Ca2+ and with the marked, but not total, reduction in Ca2+-dependent phosphorylation of endogenous substrates seen in Fig. 4. The time course of the Ca2+-induced reduction of CaMK II activity in permeabilized islets (Fig. 5) is similar to that of the transient Ca2+-induced insulin secretory response from permeabilized islets (Figs. 1 and 2).


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Fig. 5.   Time course of Ca2+-induced loss of islet CaMK II activity. Electrically permeabilized islets were incubated for 1-30 min in the presence of 50 nM Ca2+ (bullet ) or 10 µM Ca2+ (black-lozenge ), and CaMK II activity of islet extracts was estimated by phosphorylation of the exogenous substrate autocamtide-2. Islets incubated in the presence of 50 nM Ca2+ showed sustained CaMK II activity (P > 0.8 by ANOVA), whereas there was a time-dependent decrease in CaMK II activity in islets exposed to 10 µM Ca2+ (P < 0.001 by ANOVA). Points show means ± SE; n = 4.

The Ca2+-induced reduction in Ca2+/calmodulin-dependent protein kinase activity was independent of the activation of CaMK II during the preincubation period, since it also occurred when the CaMK II inhibitor CaMK-(290---309) was present during the preincubation period (Fig. 4, lanes 5 and 6). These observations suggest that the reductions in Ca2+/calmodulin-dependent protein phosphorylation in Ca2+-desensitized islets reflect a loss of CaMK II activity, and this reduction in activity cannot be explained by the activation, autophosphorylation, and consequent Ca2+ independence of CaMK II during the preincubation period.

Effects of inhibitors of phosphoprotein phosphatases and proteases. In further experiments, we investigated whether the Ca2+-induced inhibition of Ca2+/calmodulin-dependent protein phosphorylation could be accounted for by the activation of phosphoprotein phosphatases 1 (PP1), 2A (PP2A), or 2B (PP2B) or by the proteolytic inactivation of Ca2+/calmodulin-dependent kinase. Inhibition of islet phosphoserine/threonine phosphatase activities using a combination of inhibitors of PP1 and PP2A (1 µM each of CAL, CAN, and OA) and an inhibitor of PP2B (1 µM CM) enhanced the overall levels of 32P incorporation in islet extracts but had no effect on Ca2+/calmodulin-dependent protein kinase activity in islet extracts. Thus, as shown in Table 1, Ca2+/calmodulin-dependent 32P incorporation into the 54-kDa substrate was greatly reduced in extracts prepared from Ca2+-desensitized islets (incubated with 10 µM Ca2+ for 30 min), and this was not prevented by inhibition of phosphoprotein phosphatase activity in permeabilized islets during the incubation period and in the subsequent assay of Ca2+/calmodulin-dependent protein kinase activity. Similarly, the presence of a cocktail of protease inhibitors (20 µM each of E-64, PMSF, and TLCK; 50 µg/ml leupeptin; 100 µg/ml E-64C) during the incubation, extraction, and kinase assay did not prevent the Ca2+-induced loss of Ca2+/calmodulin-dependent 32P incorporation, as shown in Table 1.

                              
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Table 1.   Inhibitors of phosphoprotein phosphatases or proteases do not prevent the Ca2+-dependent reduction in Ca2+/calmodulin-dependent kinase activity in islet extracts

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ca2+ is generally considered to be the most important intracellular regulator of nutrient-induced insulin secretion from pancreatic beta -cells. Glucose causes a rapid increase in beta -cell cytosolic Ca2+ by promoting Ca2+ influx through voltage-operated Ca2+ channels, and glucose-induced insulin secretion is largely dependent on the presence of extracellular Ca2+ (reviewed in Refs. 11, 14, 36). However, although secretory responses to glucose and other nutrients are maintained for the duration of the stimulus, secretory responses to experimental elevations in intracellular Ca2+ are transient, whether studied in intact cells using Ca2+ ionophores (47) or in permeabilized beta -cell preparations (17, 19, 20). Our measurements of Ca2+-induced insulin release from electrically permeabilized islets of Langerhans in the present studies have confirmed our previous observations of the pattern of secretion (19, 20), and similar Ca2+-induced reductions in secretory responsiveness to Ca2+ have been reported in electrically permeabilized chromaffin cells (23, 24), alpha -toxin permeabilized gonadotrophs (42), and insulin-secreting HIT-T15 beta -cells (17). As we have discussed in detail elsewhere (19, 20), the loss of beta -cell secretory responsiveness to Ca2+ is not caused by time-dependent or Ca2+-dependent resealing of the permeabilized membranes or by a decrease in the overall secretory capacity of the cells, as confirmed in the present study by the secretory responses of Ca2+-insensitive beta -cells to the activation of PKC or cAMP-dependent protein kinase by PMA or cAMP, respectively.

Glucose-induced elevations in beta -cell cytosolic Ca2+ show an oscillatory pattern with rapid, small-amplitude oscillations (2-5/min) superimposed on larger-amplitude oscillations with a frequency of 0.2-0.5 oscillations/min (reviewed in Refs. 13 and 14). The physiological relevance of the slower, large-amplitude oscillations in beta -cell Ca2+ is unclear, but work in other tissues suggests that an oscillatory pattern of intracellular Ca2+ may facilitate an efficient exocytotic response, and it has also been suggested that oscillations in intracellular Ca2+ may prevent desensitization of intracellular targets to Ca2+ (12, 38). However, our measurements of insulin secretion from permeabilized islets do not provide any evidence for enhanced secretory responses to oscillatory patterns of intracellular Ca2+. Furthermore, our results suggest that the loss of responsiveness to Ca2+ was not a direct consequence of the pattern of change in cytosolic Ca2+, since essentially similar responses and time courses were observed in response to 2-min oscillations in Ca2+ and to prolonged elevations in Ca2+. These results extend our previous studies in which desensitization of Ca2+-induced insulin secretion was observed when the Ca2+ was presented as pulses of 10-min duration (20) and confirm earlier observations in alpha -toxin permeabilized HIT-T15 cells in which the secretory response to Ca2+ was not maintained by 1-min pulses of Ca2+ (17). The observation that the appearance of glucose-induced oscillations in beta -cell Ca2+ varies with species (39) may also raise questions as to the importance of the Ca2+ oscillations in the maintenance of normal secretory responses. Whatever the physiological significance of glucose-induced oscillations in beta -cell Ca2+, the transience of Ca2+-induced insulin secretion appears to be due to a selective and rapid desensitization of the secretory apparatus to Ca2+, which is unrelated to the pattern of changes in intracellular Ca2+.

Although there is no doubt about the importance of an influx of extracellular Ca2+ in the initiation of insulin secretion, the mechanisms through which elevations in intracellular Ca2+ initiate exocytosis in the beta -cell remain unclear. There is a body of circumstantial evidence that the Ca2+-dependent activation of protein kinases is an important initiator of nutrient-induced insulin secretion. Thus increases in Ca2+ cause rapid increases in protein phosphorylation in intact beta -cells and in broken cell preparations (reviewed in Refs. 2 and 10); glucose and other insulin secretagogues activate CaMK II in islets of Langerhans and in insulin-secreting cell lines (5, 31, 44), and inhibitors of CaMK inhibit depolarization-induced increases in membrane capacitance in single beta -cells (1) and inhibit glucose-induced insulin secretion from intact islets of Langerhans (29, 45). More recently, a temporal correlation between glucose-induced CaMK II activation and insulin secretion has been established and presented as evidence of a role for CaMK II in transducing secretory responses to Ca2+-mobilizing stimuli (5).

In accordance with an important role for kinase activation in beta -cell secretory responses to Ca2+, we have demonstrated previously that elevations in cytosolic Ca2+ enhanced 32P incorporation into several proteins in electrically permeabilized islets (21) and that this Ca2+-dependent protein phosphorylation was much reduced by prior exposure of the permeabilized islets to stimulatory concentrations of Ca2+ (20). These studies suggested that the desensitization of the secretory process to Ca2+ may be caused by reduced Ca2+-dependent protein phosphorylation in desensitized beta -cells but did not define the underlying cellular event. In the present studies, we have demonstrated that the Ca2+-induced reduction in Ca2+-dependent phosphorylation of endogenous substrates seen in our previous studies reflects a loss of endogenous Ca2+/calmodulin-dependent kinase activity. Furthermore, the use of a peptide substrate specific for CaMK II (9) indicates that the loss of Ca2+-induced phosphorylation is primarily due to a reduction in the phosphorylating activity of CaMK II, an enzyme widely implicated in Ca2+-induced secretory responses (5, 29, 31, 44, 45). The time course of the Ca2+-induced reductions in CaMK II activity were remarkably similar to the transient patterns of insulin secretion induced by elevations in intracellular Ca2+, with both CaMK II activity and Ca2+-induced insulin secretion being markedly reduced after 15 min exposure to a stimulatory concentration of Ca2+. These observations are fully consistent with CaMK II activity being required for insulin secretion from beta -cells.

The mechanism(s) underlying the Ca2+-induced reduction in CaMK II activity is not clear. It cannot be accounted for by a Ca2+-dependent loss of endogenous calmodulin, since the pores formed in electrically permeabilized cells are of insufficient dimensions to allow loss of endogenous proteins of the size of calmodulin (20). In any case, the concentration of calmodulin was not rate limiting in the in vitro assays for CaMK II, since an excess of exogenous calmodulin was supplied in the reaction mixture.

CaMK II is well known to autophosphorylate upon activation, reducing the Ca2+ dependence of its catalytic activity (5, 31, 37, 44), and autophosphorylated CaMK II is almost certainly one of the major endogenous substrates detected in the present studies. There are a number of reasons why the Ca2+-induced reduction in CaMK II activity is unlikely to be caused by autophosphorylation of the kinase. First, we have previously demonstrated that the loss of activity is not dependent on the presence of ATP during the preincubation period (20), whereas autophosphorylation is ATP dependent. Second, there was no corresponding increase in Ca2+-independent phosphorylations or kinase activity in Ca2+-desensitized islets, which would be expected if CaMK II were autophosphorylated and hence Ca2+ independent (44). Third, the loss of CaMK II activity was not prevented by the presence of a selective peptide inhibitor of CaMK II (32). Electrically permeabilized islets are a useful experimental model for using peptide inhibitors, and we have shown that autoinhibitory peptides will enter the permeabilized cells and selectively inhibit the appropriate kinase (3). In the present study, the autoinhibitory sequence CaMK-(290---309) was shown to inhibit CaMK activity in islet extracts but was without effect on the process of Ca2+-induced reduction of Ca2+-dependent kinase activity. These observations are supported by the report that Ca2+ desensitization of insulin secretion from permeabilized HIT-T15 beta -cells was not inhibited by KN-62, an inhibitor of CaMK II (17). Finally, the peptide substrate used in our in vitro assays, autocamtide-2, is a substrate for both the unphosphorylated, Ca2+/CaM-dependent form of CaMK II and for the autophosphorylated Ca2+-independent form of CaMK II, which is generated in islets in response to Ca2+-mobilizing stimuli (5, 39). The observed reductions in autocamtide-2 phosphorylation by extracts prepared from permeabilized islets after exposure to micromolar amounts of Ca2+ cannot therefore be explained by Ca2+-induced autophosphorylation of CaMK II, and there must exist another Ca2+-dependent mechanism through which CaMK II activity is decreased.

Pancreatic beta -cells express a number of phosphoprotein phosphatase activities, including PP1, PP2A, and PP2B (28), and there is some evidence that these enzymes may be involved in the regulation of insulin secretion by modifying the phosphorylation state of as yet unidentified proteins (7, 28). Our results suggest that the Ca2+-induced inhibition of Ca2+-dependent protein phosphorylation and of CaMK II activity was not dependent on the activation of PP1, PP2A, or PP2B in the permeabilized islets, since the loss of Ca2+/calmodulin-dependent kinase activity was not prevented by the presence of inhibitors of these enzymes during the desensitization period and the subsequent kinase assay. An additional class of phosphoprotein phosphatase, phosphoprotein phosphatase 2C (PP2C), has been reported to dephosphorylate recombinant autophosphorylated CaMK II, thus restoring its Ca2+ dependency (8). However, as discussed above, the Ca2+-induced loss of CaMK II activity cannot be accounted for by changes in the phosphorylation state and thus Ca2+ dependence of the enzyme. Furthermore, although we cannot rule out a role for PP2C in the generation of Ca2+ desensitization in beta -cells, PP2C expression in pancreatic beta -cells has not yet been confirmed, and the ionic dependence of PP2C on Mg2+ rather than Ca2+ makes it an unlikely candidate for a Ca2+-dependent process.

An alternative Ca2+-dependent but calmodulin- and ATP-independent mechanism for the reduction in Ca2+/calmodulin-dependent kinase activity could be through the activation of beta -cell proteases, and there is some evidence that this mechanism is operational for other protein kinase activities in beta -cells. Thus the family of diacylglycerol-sensitive, Ca2+/phospholipid-dependent PKC isoenzymes is susceptible to proteolytic cleavage by membrane-associated Ca2+-activated proteases (30). This may be the basis for the reduction in beta -cell PKC activity by prolonged activation of the enzyme (15), since the loss of enzyme activity in permeabilized islets was reduced by the presence of the protease inhibitors leupeptin and PMSF (35). The calpains are Ca2+-dependent cysteine proteases, which are ubiquitously expressed in mammalian cells (reviewed in Ref. 27) and which are known to proteolyse many calmodulin-binding proteins, including several protein kinases (reviewed in Ref. 43). However, in our study, the Ca2+-induced reduction in Ca2+/calmodulin-dependent kinase activity was not prevented by the presence of a mixture of protease inhibitors, which are known to inhibit calpain activity (26) and which would have ready access to the enzyme in the electrically permeabilized islets used in our experiments. These results suggest that the reduction in Ca2+/calmodulin-dependent kinase activity was not caused by the activation of Ca2+-dependent proteases, and the mechanism underlying the desensitization process in beta -cells remains unclear.

The importance of elevations in intracellular Ca2+ in the generation and maintenance of physiologically appropriate insulin secretory responses is well established (reviewed in Refs. 11, 13, 14, 36). It is not yet clear whether the Ca2+ desensitization observed in the current studies occurs under more physiological conditions, although it has been suggested that disturbances in beta -cell Ca2+ handling may be involved in the secretory defects seen in type 2 diabetes mellitus (12). Irrespective of its function in pathological conditions, our experimental observations support a role for CaMK II in the regulation of insulin secretion. Thus Ca2+-mobilizing insulin secretagogues promote rapid increases in CaMK II activity in islets (5), and insulin secretion is inhibited by inhibitors of CaMK II (29, 45). The present demonstration of reduced CaMK II activity concomitant with reduced secretory responsiveness to Ca2+ provides further evidence that the activation of Ca2+/calmodulin-dependent kinase(s) is important in the generation of appropriate insulin secretory responses to the elevations in intracellular Ca2+, which are induced by physiologically important nutrient secretagogues, such as glucose.

    ACKNOWLEDGEMENTS

Financial support from the Medical Research Council and the Wellcome Trust is gratefully acknowledged.

    FOOTNOTES

S. J. Persaud was a Wellcome Trust Research Fellow (Grant no. 039057).

Address for reprint requests: P. Jones, Biomedical Sciences Division, King's College London, Campden Hill Rd., London W8 7AH, UK.

Received 11 June 1997; accepted in final form 18 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Endocrinol Metab 274(4):E708-E715
0193-1849/98 $5.00 Copyright © 1998 the American Physiological Society




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