Glucagon-like peptide 1 activates protein kinase C through Ca2+-dependent activation of phospholipase C in insulin-secreting cells

Although the stimulatory effect of glucagon-like peptide 1 (GLP-1), a cAMP-generating agonist, on Ca(2+) signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca(2+) influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca(2+) signaling can activate PKCalpha and PKCepsilon at a substimulatory concentration of glucose (3 mm) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKCalpha and PKCepsilon from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca(2+)-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKCepsilon inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCepsilon-(149-164) (antp-PKCepsilon) and a conventional PKC inhibitor, G?-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKCgamma and 2) translocation of PKCalpha-DsRed and PKCepsilon-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKCalpha and PKCepsilon, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.     

Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Ca(2+)-induced Ca2+ release in insulin-secreting cells.

The sulphydryl reagent thimerosal (50 microM) released Ca2+ from a non-mitochondrial intracellular Ca2+ pool in a dose-dependent manner in permeabilized insulin-secreting RINm5F cells. This release was reversed after addition of the reducing agent dithiothreitol. Ca2+ was released from an Ins(1,4,5)P3-insensitive pool, since release was observed even after depletion of the Ins(1,4,5)P3-sensitive pool by a supramaximal dose of Ins(2,4,5)P3 or thapsigargin. The Ins(1,4,5)P3-sensitive pool remained essentially unaltered by thimerosal. Thimerosal-induced Ca2+ release was potentiated by caffeine. These findings suggest the existence of Ca(2+)-induced Ca2+ release also in insulin-secreting cells.     

Capacitative Ca2+ entry involves Ca2+ influx factor in rat glioma C6 cells.

Capacitative Ca2+ entry exists in rat glioma C6 cells; however, how the information of depletion of Ca2+ in intracellular stores transmits to the plasma membrane is unknown. In the present study, we examined whether Ca2+ influx factor (CIF) causes capacitative Ca2+ entry in C6 cells. CIF was extracted from non-treated (Non-CIF), bombesin-treated (BBS-CIF) and thapsigargin-treated (TG-CIF) C6 cells by a reverse-phase silica cartridge. The addition of BBS-CIF and TG-CIF gradually increased cytoplasmic Ca2+ concentration ([Ca2+]i) but Non-CIF did not increase [Ca2+]i. Neither BBS-CIF nor TG-CIF elevated [Ca2+]i in the absence of extracellular Ca2+. Gd3+ inhibited the increase in [Ca2+]i induced by BBS-CIF and TG-CIF. Genistein abolished an elevation of [Ca2+]i induced by BBS-CIF and TG-CIF. BBS-CIF and TG-CIF did not increase inositol 1,4,5-trisphosphate accumulation. The results suggest that capacitative Ca2+ entry is caused by CIF in rat glioma C6 cells.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.     

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).     

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor

Thecoupling mechanism between depletion of Ca²⁺ stores in theendoplasmic reticulum and plasma membrane store-operated ion channelsis fundamental to Ca²⁺ signaling in many cell types and hasyet to be completely elucidated. Using Ca²⁺release-activated Ca²⁺ (CRAC) channels in RBL-2H3 cells asa model system, we have shown that CRAC channels are maintained in theclosed state by an inhibitory factor rather than being opened by theinositol 1,4,5-trisphosphate receptor. This inhibitory role can befulfilled by the Drosophila protein INAD (inactivation-noafter potential D). The action of INAD requires Ca²⁺ andcan be reversed by a diffusible Ca²⁺ influx factor. Thusthe coupling between the depletion of Ca²⁺ stores and theactivation of CRAC channels may involve a mammalian homologue of INADand a low-molecular-weight, diffusible store-depletion signal.

     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

Real-time analysis of phospholipase C activity during different patterns of receptor-induced Ca2+ responses in HEK293 cells

[Ca(2+)](i) oscillations can either depend on oscillatory inositol-1,4,5-trisphosphate (InsP(3)) formation by phospholipase C (PLC) or rely on local feedback mechanisms involving the InsP(3) receptor. To assess the PLC activity underlying carbachol-induced [Ca(2+)](i) oscillations in single HEK293 cells, we co-imaged [Ca(2+)](i) with fluorescent fusion proteins of protein kinase C (PKC) isotypes and the PH domain of PLC-delta 1 (PLC-delta 1(PH)). The translocation of PKC alpha-YFP in single cells followed two discrete patterns. Upon maximally effective agonist concentrations, a fast association and delayed dissociation (k(on)>k(off)) was the predominant pattern. The delayed dissociation has been linked to diacylglycerol formation. Upon stimulation with submaximally effective agonist concentrations as well as during regenerative [Ca(2+)](i) waves, we mainly observed short translocations with k(on) approximately equal to k(off). Translocation time courses and efficiencies of the diacylglycerol-sensing PKC epsilon-CFP and the InsP(3)/phosphatidylinositol-4,5-bisphosphate-sensing YFP-PLC-delta 1(PH) were closely correlated. Significant PLC activity was only detectable upon strong receptor stimulation, which typically failed to trigger [Ca(2+)](i) oscillations. During [Ca(2+)](i) oscillations induced by submaximal receptor stimulation, YFP-PLC-delta 1(PH) did not translocate, whereas a fluorescent PKC epsilon fusion protein has been reported to exhibit a slow, non-oscillatory accumulation at the plasma membrane. We conclude that carbachol-induced [Ca(2+)](i) oscillations in HEK293 cells develop at low levels of presumably non-oscillatory PLC activity.     

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

Growth hormone promotes Ca(2+)-induced Ca2+ release in insulin-secreting cells by ryanodine receptor tyrosine phosphorylation

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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Vasopressin directly closes ATP-sensitive potassium channels evoking membrane depolarization and an increase in the free intracellular Ca2+ concentration in insulin-secreting cells.

The effects of arginine-vasopressin (AVP) (0.01-1 microM) on membrane potential, [Ca2+]i and ATP-sensitive potassium channels have been studied in the insulin-secreting cell line RINm5F. In whole cells, with an average spontaneous cellular transmembrane potential of -64 +/- 3 mV (n = 33) and an average basal [Ca2+]i of 102 +/- 6 nM (n = 40), AVP evoked: (i) membrane depolarization, (ii) voltage-dependent Ca2+ spike-potentials and (iii) a sharp rise in [Ca2+]i. Single-channel current events recorded from excised outside-out membrane patches show that AVP closes potassium channels that are also closed by tolbutamide (100 microM) and opened by diazoxide (100 microM). AVP acts on KATP channels specifically from the outside of the membrane and a soluble cytosolic messenger appears not to be involved, since there is no channel activation in cell-attached membrane patches when the peptide is added to the bath solution.