Omission Of Extracellular Ca2 Research Articles

Effects of Spirulina platensis on insulin secretion, dipeptidyl peptidase IV activity and both carbohydrate digestion and absorption indicate potential as an adjunctive therapy for diabetes.

Spirulina platensis has been found to be useful in the treatment of type 2 diabetes. The present study aims to elucidate the effects of ethanol extract and butanol fraction of S. platensis on insulin release and glucose homoeostasis in type 2 diabetic rats, together with their mechanism of actions. In vitro and in vivo methods were used including cellular studies to determine potential role of ion channels and cAMP in the insulinotropic actions of the extracts. The ethanol extract and butanol fraction stimulated insulin release from mouse islets and pancreatic β-cells in a concentration-dependent manner. The butanol fraction also similarly stimulated insulin release from perfused rat pancreas. The insulin-releasing action was augmented by glucose, isobutylmethylxanthine, tolbutamide and a depolarising concentration of KCl. The insulin secretory effect was attenuated with diazoxide and verapamil and by omission of extracellular Ca2+. Butanol fraction was found to significantly inhibit dipeptidyl peptidase IV enzyme activity. Moreover, butanol fraction improved glucose tolerance following oral glucose administration (2·5 g/kg body weight (b.w.)). The butanol fraction was tested on 24 h starved rats given an oral sucrose load (2·5 g/kg b.w.) to examine possible effects on carbohydrate digestion and absorption. S. platensis substantially decreased postprandial hyperglycaemia after oral sucrose load and increased unabsorbed sucrose content throughout the gut. During in situ intestinal perfusion with glucose, the butanol fraction reduced glucose absorption and promoted gut motility. Finally, chronic oral administration of butanol fraction for 28 d significantly decreased blood glucose, increased plasma insulin, pancreatic insulin stores, liver glycogen and improved lipid profile. The characterisation of active compounds from butanol fraction revealed the presence of p-coumaric acid, β-carotene, catechin and other antioxidant polyphenols. In conclusion, S. platensis could be an adjunctive therapy for the management of type 2 diabetes.

ATP-Evoked Intracellular Ca²⁺ Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

Role of Calcium in Glucagon Secretion

The role of calcium ions in the process by which nutrients affect glucagon secretion by pancreatic islet α-cells remains the matter of an apparently endless debate. In the prolongation of recent articles dealing with this matter, the present review draws attention to the dual role of Ca2+ as revealed by prior publications. In such a perspective, emphasis is placed on the increase in glucagon output in response to the omission of extracellular Ca2+ as recorded in the presence of D-glucose or 2-ketoisocaproate, the permissive role of extracellular Ca2+ in the positive secretory response to arginine or a mixture of fumarate, glutamate and pyruvate, and the effects of an organic calcium-antagonist on glucagon output. Considering the role currently ascribed to Ca2+ in the activation of motile events involved in stimulus-secretion coupling, attention is also given to the effects of cytochalasin B, D2O and mitotic-spindle inhibitors upon the secretory response of α-cells exposed to D-glucose in the absence or presence of arginine.

Bombesin facilitates GABAergic transmission and depresses epileptiform activity in the entorhinal cortex

Bombesin and the bombesin-like peptides including neuromedin B (NMB) and gastrin-releasing peptide (GRP) are important neuromodulators in the brain. We studied their effects on GABAergic transmission and epileptiform activity in the entorhinal cortex (EC). Bath application of bombesin concentration-dependently increased both the frequency and amplitude of sIPSCs recorded from the principal neurons in the EC. Application of NMB and GRP exerted the same effects as bombesin. Bombesin had no effects on mIPSCs recorded in the presence of TTX but slightly depressed the evoked IPSCs. Omission of extracellular Ca(2+) or inclusion of voltage-gated Ca(2+) channel blockers, Cd(2+) and Ni(2+), blocked bombesin-induced increases in sIPSCs suggesting that bombesin increases GABA release via facilitating extracellular Ca(2+) influx. Bombesin induced membrane depolarization and slightly increased the input resistance of GABAergic interneurons recorded from layer III of the EC. The action potential firing frequency of the interneurons was also increased by bombesin. Bombesin-mediated depolarization of interneurons was unlikely to be mediated by the opening of a cationic conductance but due to the inhibition of inward rectifier K(+) channels. Bath application of bombesin, NMB and GRP depressed the frequency of the epileptiform activity elicited by deprivation of Mg(2+) from the extracellular solution suggesting that bombesin and the bombesin-like peptides have antiepileptic effects in the brain.

Transient Receptor Potential Melastatin 2 Is Required for Lipopolysaccharide-Induced Cytokine Production in Human Monocytes

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable nonselective cation channel that is stimulated by oxidative stress and specifically activated by intracellular ADP-ribose. Because TRPM2 is highly expressed in immunocytes, a role of this channel in inflammation processes has been proposed. The aim of the current study was to determine the function of TRPM2 in LPS-induced cytokine production of human monocytes. Incubation of human primary monocytes with LPS resulted in an upregulation of TRPM2 mRNA, protein, and of ADP-ribose-induced membrane currents. By using short hairpin RNA to downregulate TRPM2 expression in THP-1 monocytes, we demonstrate that TRPM2 is required for the LPS-induced production of IL-6, IL-8, IL-10, and TNF-alpha. Application of LPS led to a time-dependent increase in intracellular Ca(2+) concentrations in THP-1 cells that was clearly reduced by downregulation of TRPM2. Omission of extracellular Ca(2+) strongly decreased TNF-alpha production in TRPM2-expressing cells. Thus, TRPM2-mediated Ca(2+) entry is a central mechanism for LPS-induced cytokine production in monocytic cells. The identification of TRPM2 as a major player in this LPS-dependent process makes it a promising tool in modulating monocyte functions.

Doxorubicin-induced vasomotion and [Ca(2+)](i) elevation in vascular smooth muscle cells from C57BL/6 mice.

To explore the action of doxorubicin on vascular smooth muscle cells. Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca(2+)](i) in isolated aortic smooth muscle cells was measured by using Fluo-3. Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca(2+)](i) in single muscle cells. Treatment with 10 micromol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca(2+) or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 micromol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 micromol/L increased the frequency of the RC only. In the presence of 100 micromol/L caffeine, however, 100 micromol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 micromol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca(2+)-free solution, doxorubicin induced a transient [Ca(2+)](i) increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca(2+)](i) was suppressed by nifedipine and potentiated by ryanodine and charybdotoxin. Doxorubicin not only releases Ca(2+) from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca(2+) into vascular smooth muscle cells.

Stimulated Efflux of Amino Acids and Glutathione from Cultured Hippocampal Slices by Omission of Extracellular Calcium: LIKELY INVOLVEMENT OF CONNEXIN HEMICHANNELS

Omission of extracellular Ca(2+) for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X(7) receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca(2+) omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca(2+) omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca(2+) and Mg(2+) were omitted from the medium. Omission of Ca(2+) for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca(2+) stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.

Monensin causes transient calcium ion influx into mouse splenic lymphocytes in a sodium ion-independent fashion

Monensin, a Na + ionophore, can increase cytosolic free Ca 2+ concentration ([Ca 2+] i) in many cell types, but no studies have investigated the mechanism underlying a monensin-induced increase in [Ca 2+] i in immune cells. In view of this, we investigated the effect of monensin on [Ca 2+] i and cytosolic free Na + concentration ([Na +] i) in mouse splenic lymphocytes using a fluorescence Ca 2+ indicator, fura-2, and a fluorescence Na + indicator, sodium-binding benzofuran isophthalate (SBFI), respectively. Monensin (1–100 μM) caused transient and sustained increases in [Ca 2+] i and [Na +] i, respectively, in a concentration-dependent manner. The monensin-induced increase in [Ca 2+] i was abolished by the omission of extracellular Ca 2+ or 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100–150 μM), and was largely inhibited by Ni 2+ (2–5 mM). The omission of extracellular Na + failed to inhibit the monensin-induced increases in [Ca 2+] i. Furthermore, tetrodotoxin (1–10 μM), 5-( N, N-dimethyl)-amiloride (DMA, 10–20 μM), 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400, 3–10 μM), verapamil (10–200 μM), nifedipine (10–200 μM), ω-agatoxin IVA (0.2–10 μM), ω-conotoxin GVIA (1–10 μM), ω-conotoxin MVIIC (0.5–10 μM), and nordihydroguaiaretic acid (NDGA, 1–10 μM) had no effect on the increases in [Ca 2+] i. Monensin-induced Mn 2+ influx into splenic lymphocytes. The Mn 2+ influx was completely inhibited by SKF-96365. These results suggest that monensin transiently increases [Ca 2+] i in mouse splenic lymphocytes by stimulating Ca 2+ entry via non-selective cation channels in a Na +-independent manner.

Influx of extracellular Ca2+ is necessary for electrotaxis inDictyostelium

Intracellular free Ca2+ ([Ca2+](i)) is a pivotal signalling element in cell migration and is thought to be required for chemotaxis of Dictyostelium. Ca2+ signalling may also be important for electrotaxis. However this suggestion has been controversial. We show that electric fields direct Dictyostelium cells to migrate cathodally and increase [Ca2+](i) in Dictyostelium cells, as determined by Fluo-3 AM imaging and (45)Ca2+ uptake. Omission of extracellular Ca2+([Ca2+](e)) and incubation with EGTA abolished the electric-field-stimulated [Ca2+](i) rise and directional cell migration. This suggests a requirement for [Ca2+](e) in the electrotactic response. Deletion of iplA, a gene responsible for chemoattractant-induced [Ca2+](i) increase, had only a minor effect on the electric-field-induced [Ca2+](i) rise. Moreover, iplA-null Dictyostelium cells showed the same electrotactic response as wild-type cells. Therefore, iplA-independent Ca2+ influx is necessary for electrotactic cell migration. These results suggest that the [Ca2+](i) regulatory mechanisms induced by electric fields are different from those induced by cAMP and folic acid in Dictyostelium cells. Different roles of the iplA gene in chemoattractant-induced and electrically induced Ca2+ signalling, and different effects of [Ca2+](i) elevation on chemotaxis and electrotaxis indicate that the chemoattractant and electric cues activate distinctive initial signalling elements.

Group III metabotropic glutamate receptor activation inhibits Ca2+ influx and nitric oxide synthase activity in bone marrow stromal cells

Nitric oxide (NO) is pivotal to bone physiology. In the central nervous system constitutive, Ca(2+)-calmodulin regulated NO synthase activity and glutamate signalling are intimately linked. Since L-glutamate signalling occurs in bone and is implicated in bone regulation, we have investigated the effect of L-glutamate on NO synthase in bone-derived cells. Treatment of marrow stromal cells with L-glutamate reduced basal NO synthase activity by 40%. Imaging showed that L-glutamate caused a rapid, usually localised and slowly-reversible fall in [Ca(2+)](i). This effect was resistant to disruption of intracellular Ca(2+) stores but sensitive to extracellular La(3+) or omission of extracellular Ca(2+), demonstrating that glutamate acts by inhibition of membrane Ca(2+) influx. The only previous description of such an effect of L-glutamate is via activation of the group III receptor, mGluR6, in the retina. Using Western blotting and RT-PCR we detected mGluR6 protein and transcripts in marrow stromal cells. The effects of L-glutamate on NOS activity and [Ca(2+)](i) in marrow stromal cells were abolished by a group III mGluR inhibitor, (S)-2-amino-2-methyl-4-phosphonobutyric acid. Recording of membrane potential showed that, similarly to the effects of retinal mGluR6 activation, L-glutamate induced membrane hyperpolarisation (-16 +/- 2 mV), which was also sensitive to group III mGluR inhibition. L-glutamate had no effect on cAMP levels. We conclude that activation of a group III mGluR in bone marrow stromal cells inhibits a Ca(2+)-permeable plasma membrane channel, reducing [Ca(2+)](i) and suppressing generation of NO. These observations directly link bone L-glutamate signalling to processes central to bone growth and regulation.

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

There is little information available concerning the link between the ryanodine (RY) receptors and the downstream Ca(2+) signaling events in beta-cells. In fura-2 loaded INS-1E cells, activation of RY receptors by 9-methyl 5,7-dibromoeudistomin D (MBED) caused a rapid rise of [Ca(2+)]i followed by a plateau and repetitive [Ca(2+)]i spikes on the plateau. The [Ca(2+)]i plateau was abolished by omission of extracellular Ca(2+) and by SKF 96365. In the presence of SKF 96365, MBED produced a transient increase of [Ca(2+)]i, which was abolished by thapsigargin. Activation of RY receptors caused Ca(2+) entry even when the ER Ca(2+) pool was depleted by thapsigargin. The [Ca(2+)]i plateau was not inhibited by nimodipine or ruthenium red, but was inhibited by membrane depolarization, La(3+), Gd(3+), niflumic acid, and 2-aminoethoxydiphenyl borate, agents that inhibit the transient receptor potential channels. The [Ca(2+)]i spikes were inhibited by nimodipine and ryanodine, indicating that they were due to Ca(2+) influx through the voltage-gated Ca(2+) channels and Ca(2+)-induced Ca(2+) release (CICR). Activation of RY receptors depolarized membrane potential as measured by patch clamp. Thus, activation of RY receptors leads to coherent changes in Ca(2+) signaling, which includes activation of TRP-like channels, membrane depolarization, activation of the voltage-gated Ca(2+) channels and CICR.

PROPYLENE GLYCOL INCREASES CYTOSOLIC FREE CALCIUM IN RAT CEREBROCORTICAL SYNAPTOSOMES

In these studies, the authors investigated the effect of propylene glycol (PG) on the cytosolic free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes using the fluorescent Ca2+ indicator fura-2. PG (0.5-5% v/v) increased [Ca2+]i in a concentration-dependent manner. The PG-induced increase in [Ca2+]i was inhibited approximately 50% by the omission of extracellular Ca2+ or the addition of Ni2+ (100 μM). Decrease of extracellular Na+ (6.2 mM) or addition of tetrodotoxin (1 μM), verapamil (10 μM), nifedipine (10 μM), ω-agatoxin IVA (200 nM), ω-conotoxin GVIA (1 μM), or ω-conotoxin MVIIC (1 μM) had no effect on the increase in [Ca2+]i. Also, addition of TMB-8 (100 μM), ryanodine (50 μM) or thapsigargin (1 μM) did not modify the increase in [Ca2+]i in the absence of extracellular Ca2+. These results suggest that PG increases [Ca2+]i in rat cerebrocortical synaptosomes by both stimulat-ing Ca2+ entry through a Ni2+-sensitive pathway and releasing Ca2+ from TMB-8-, ryanodine- and thapsigargin-insensitive Ca2+ stores

Copper-induced formation of reactive oxygen species causes cell death and disruption of calcium homeostasis in trout hepatocytes.

We have previously shown that copper is acutely toxic for trout hepatocytes, inducing enhanced influx of Ca(2+) and a loss of cell viability. The aim of the present study was to elucidate the pathways of Ca(2+) entry into the cells, the hypothetical role of reactive oxygen species (ROS) in copper toxicity, and the interaction of ROS formation and the disruption of Ca(2+) homeostasis. We found that, acutely, copper-induced cell death occurred independently from an increase of intracellular free Ca(2+) (Ca(2+)i), but could be prevented by addition of agents interfering with ROS production. Addition of the Ca(2+) channel blocker verapamil did not affect the Ca(2+)i increase evoked by copper, whereas in the presence of LaCl(3), an inhibitor of both Ca(2+) channels and Na(+)/Ca(2+)-exchange, this increase was significantly delayed. ROS formation, estimated by use of the fluorescence indicator 2',7'-dichlorofluorescin diacetate, was significantly enhanced by copper. Omission of extracellular Ca(2+) or addition of either verapamil or LaCl(3) did not diminish ROS formation induced by copper. In contrast, the hydroxyl radical scavenger dimethyl sulfoxide and the ferric ion chelator deferoxamine inhibited radical production. In addition, these agents either partially (dimethyl sulfoxide) or completely (deferoxamine) prevented an increase of Ca(2+)i. Altogether our results indicate that ROS formation is the crucial event leading to cell death during acute exposure to copper, whereas the increase of Ca(2+)i is a secondary, acutely less toxic, phenomenon. Furthermore, these findings suggest that Ca(2+) entry occurs via a LaCl(3)-sensitive pathway, presumably representing Na(+)/Ca(2+)-exchange, and non-specific membrane leaks induced by lipid peroxidation in the presence of copper.

Palytoxin-induced increase in cytosolic-free Ca 2+ in mouse spleen cells

The effect of palytoxin (C 129H 223N 3O 54) on Ca 2+ homeostasis in immune cells has not been studied. Therefore, we investigated the effect of palytoxin on the cytosolic-free Ca 2+ concentration ([Ca 2+] i ) in mouse spleen cells using a fluorescence Ca 2+ indicator, fura-2. Palytoxin (0.1–100 nM) increased [Ca 2+] i in a concentration-dependent manner. The palytoxin-induced increase in [Ca 2+] i was abolished by the omission of extracellular Ca 2+ or 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100 μM), and was greatly inhibited by Ni 2+ (2 mM). Ouabain (0.5–1 mM) partially inhibited the palytoxin-induced response. There was no effect of decreased extracellular Na + (6.2 mM), tetrodotoxin (1 μM), verapamil (10 μM), nifedipine (10 μM), ω-agatoxin IVA (200 nM), ω-conotoxin GVIA (1 μM), ω-conotoxin MVIIC (500 nM), or La 3+ (100 μM). These results suggest that palytoxin increases [Ca 2+] i in mouse spleen cells by stimulating Ca 2+ entry through an SKF-96365-, Ni 2+-sensitive pathway.

Pharmacology of ischemia-induced glutamate efflux from rat cerebral cortex in vitro

Simulated ischemic conditions (hypoxia–hypoglycaemia) in vitro enhanced glutamate efflux from rat cerebrocortical prisms. Here we characterised efflux mechanisms using pharmacological tools. The Na + channel blocker TTX (1 μM) did not affect ischemia-induced efflux, while sipatrigine (100 μM), a Na +/Ca 2+ channel blocker and ω-conotoxin MVIIC (2 μM), an N/P/Q type Ca 2+ channel blocker, inhibited efflux by fractions of 0.53 and 0.46, respectively (1.00 corresponding to total inhibition). Omission of extracellular Ca 2+ and addition of EGTA (2 mM) inhibited ischemia-induced efflux only during the first 25 min of incubation. A similar result was observed on omission of extracellular Ca 2+ together with addition of La 3+ (10 μM) and Mg 2+ (6 mM). TTX, sipatrigine and La 3+/Mg 2+ all inhibited control efflux. Ischemia-induced efflux was sensitive to the volume activated anion channel inhibitor NPPB (100 μM) only after the first 25 min of incubation, with the maximal fraction inhibited being 0.54. The glutamate transporter inhibitor d,l-TBOA reduced ischemia-induced efflux throughout a 45-min incubation period, and enhanced efflux from control tissue. d,l-TBOA inhibited efflux at 30 min by a maximum fraction of 0.49, at 50 μM. These data indicate that the early phase of ischemia-induced glutamate efflux is in part Ca 2+ dependent, while the later phase involves volume activated anion currents and both phases involve excitatory amino acid transporters.

Protein kinase A translocation and insulin secretion in pancreatic beta-cells: studies with adenylate cyclase toxin from Bordetella pertussis.

Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the beta-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting betaTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca(2+) totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca(2+), which were blocked by nifedipine and the omission of extracellular Ca(2+). Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca(2+). PKA alpha, beta and gamma catalytic subunits (Calpha, Cbeta and Cgamma respectively) were identified in betaTC6 cells by confocal microscopy. Glucose and glucagon-like polypeptide-1 (GLP-1) caused translocation of Calpha to the nucleus and of Cbeta to the plasma membrane and the nucleus, but did not affect the distribution of Cgamma. In conclusion, glucose and GLP-1 amplify insulin secretion via cAMP production and PKAbeta activation.

Sustained elevation of calcium induces Ca 2+/calmodulin-dependent protein kinase II clusters in hippocampal neurons

Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca 2+/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75–175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076–3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl- D-aspartate (250 μM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca 2+. Indeed, intracellular Ca 2+ monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca 2+. Moreover, CaMKII does not cluster when the rise in intracellular Ca 2+ is prevented by the omission of extracellular Ca 2+ during CCCP treatment, confirming that clustering is Ca 2+-dependent. A lag period of 1–2 min between the onset of high intracellular Ca 2+ levels and the formation of CaMKII clusters suggests that a sustained increase in Ca 2+ level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca 2+ overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.

Smooth Muscle Cell Response to Mechanical Injury Involves Intracellular Calcium Release and ERK1/ERK2 Phosphorylation

We have investigated possible signaling pathways coupled to injury-induced ERK1/2 activation and the subsequent initiation of vascular rat smooth muscle cell migration and proliferation. Aortic smooth muscle cells were cultured to confluency and subjected to in vitro injury under serum-free conditions. In fluo-4-loaded cells, injury induced a rapid wave of intracellular Ca2+ release that propagated about 200 μm in radius from the injured zone, reached a peak in about 20 s, and subsided to the baseline within 2 min. The wave was abolished by prior treatment with the sarcoplasmic reticulum ATPase inhibitor thapsigargin, but not by omission of extracellular Ca2+. ERK1/2 activation reached a peak at 10 min after injury and was inhibited by the MEK1 inhibitor PD98059, as well as by thapsigargin, fluphenazine, genistein, and the Src inhibitor PP2. These inhibitors also reduced [3H]thymidine incorporation and migration of cells into the injured area determined at 48 h after injury. These results show that mechanical injury to vascular smooth muscle cells induces a Ca2+ wave which is dependent on intracellular Ca2+ release. Furthermore, the injury activates ERK1/2 phosphorylation as well as cell migration and replication.

Swelling-induced changes in cytosolic [Ca 2+] in insulin-secreting cells: a role in regulatory volume decrease?

Exposure of insulin-secreting cells to hypotonic solutions causes cell swelling followed by regulatory volume decrease (RVD). We have previously demonstrated that RVD is due to activation of a Cl − conductance. The present study investigates whether changes in cytosolic [Ca 2+] play a role in these responses. Hypotonic swelling of RINm5F insulinoma cells caused a marked increase in cytosolic [Ca 2+]. This effect was abolished by omission of extracellular Ca 2+, by the Ca 2+ channel blockers D600 or Gd 3+and by 4,4′-dithiocyanatostilbene-2,2′-disulphonic acid (DIDS), an inhibitor of the volume-sensitive anion. RVD was markedly impaired in the absence of extracellular Ca 2+, but not by D600 nor by Gd 3+. RVD was also inhibited by the maxi-K + (BK Ca) channel blockers tetraethylammonium (TEA) and iberiotoxin (IbTx), whereas the K ATP channel blocker tolbutamide was ineffective. Cell swelling was accompanied by activation of a K + conductance which was sensitive to TEA and IbTx but not to tolbutamide. It is concluded that cell swelling causes activation of the volume-sensitive anion channel, leading to depolarization and Ca 2+ entry via voltage-gated Ca 2+ channels. RVD is a Ca 2+-dependent process, requiring low ‘resting’ levels of intracellular [Ca 2+]. However, the swelling-induced increase in cytosolic [Ca 2+] is not required for RVD to occur. RVD depends upon simultaneous activation of Cl − and K + channels. We suggest that the BK Ca channel is the major K + conductance involved in RVD.

The Mechanism of Maitotoxin-Induced Elevation of the Cytosolic Free Calcium Level in Rat Cerebrocortical Synaptosomes

The present study was conducted to elucidate the mechanism of the maitotoxin (MTX)-induced increase in intrasynaptosomal free calcium level ([Ca2+]i). The MTX (1 ng/ml)-induced increase in [Ca2+]i was partially inhibited by the omission of extracellular Ca2+ (Ca2+e) or the addition of verapamil, but not by adding nifedipine, w-agatoxin IVA, ω-conotoxin GVIA and w-conotoxin MVIIC. An increase in [Ca2+]i in the absence of Ca2+e was sensitive to procaine, TMB-8, genistein and verapamil, but not to ryanodine and U-73122. These results may suggest that MTX increases [Ca2+]i by stimulating Ca2+ entry through voltage-independent nonselective cation channels and Ca2+ release from stores through a phospholipase C-γl-medi-ated pathway in rat cerebrocortical synaptosomes.