Rise In Cytosolic Free Ca2 Research Articles

Measured increase in intracellular Ca 2+ during stimulated release of endogenous glutamate from human cerebrocortical synaptosomes

Presynaptic terminals (synaptosomes) prepared from guinea pig and rat cerebral cortex release endogenous glutamate in a Ca 2+-dependent manner in response to membrane depolarisation. In the present study, synaptosomes were prepared from human cerebral cortex removed in association with temporal lobe resections in epileptic patients. The cytosolic free Ca 2+ concentration increased from 474±66 before to 649±89 nM after 2 min depolarisation. The basal level of free cytosolic Ca 2+ is higher and the increase in response to depolarisation is more pronounced in human synaptosomes than observed in animal experiments. The Ca 2+-dependent glutamate release, estimated as the difference between total — and the Ca 2+-independent glutamate release, increased from 0 to 5.4±1.9 nmol/mg protein. The released amount of glutamate is larger than reported in animal models. These results demonstrate that membrane depolarisation of synaptosomes from human brain evokes a rapid rise in cytosolic free Ca 2+ and a more prolonged rise in synaptic, Ca 2+-dependent glutamate release.

Millisecond UV‐B irradiation evokes prolonged elevation of cytosolic‐free Ca2+ and stimulates gene expression in transgenic parsley cell cultures

Chalcone synthase (CHS) is a key enzyme leading to the generation of protective flavonoids in plants under environmental stress. Expression of the CHS gene is strongly upregulated by exposures to UV light, a response also observed in heterotrophic parsley cell cultures. Although there are hints that the stimulus for CHS expression may be coupled to UV-B irradiation through a rise in cytosolic-free Ca2+ ([Ca2+]i), the temporal relationship of these events has never been investigated critically. To explore this question, we have used a CHS promoter/luciferase (CHS/LUC) reporter gene fusion and recorded its expression and [Ca2+]i elevation in a transgenic parsley cell culture following millisecond light pulses. Luciferase expression was enhanced maximally seven- (+/- 2) fold by 30 10 ms flashes of UV-B light. The response was specific to wavelengths of 300-330 nm and could be inhibited in the presence of the Ca2+ channel blocker nifedipine. In parallel measurements, using Fura-2 fluorescence ratio microphotometry, we found that 10 ms UV-B flashes also evoked a gradual and prolonged rise of [Ca2+]i in the parsley cells which was irreversible within the timescale of these experiments, but could be prevented by prior treatment with nifedipine. These, and additional results, indicate a remarkably high temporal sensitivity to, and specificity for, UV-B light in CHS gene expression independent of UV-mediated DNA damage by thymine dimerization. The ability of transient UV-B stimulation to evoke prolonged elevations of [Ca2+]i suggests a functional coupling between the initial light stimulus and subsequent gene expression that takes place many tens of minutes later.

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca 2+ channels in rat pheochromocytoma cells

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [ 3H]norepinephrine ([ 3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an ic 50 value of 1.0 ± 0.2 μM. The DMPP-induced rise in cytosolic free Ca 2+ concentration [Ca 2+] i was inhibited by CPZ with an ic 50 of 1.9 ± 0.1 μM. The DMPP-induced increase in cytosolic free Na + concentration [Na +] i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [ 3H]nicotine to PC12 cells was inhibited by CPZ with an ic 50 value of 2.7 ± 0.6 μM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K +-induced [ 3H]NE secretion and [Ca 2+] i increase were inhibited by CPZ with ic 50 of 7.9 ± 1.1 and 6.2 ± 0.3 μM, respectively. Experiments with Ca 2+ channel antagonists suggest that L-type Ca 2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca 2+ channels, with the former being more sensitive to CPZ.

Modulation of glucose-induced insulin secretion by cytosolic redox state in clonal β-cells

Nutrient stimulation of pancreatic β-cells increases the cellular reduced pyridine nucleotide content, but the specific role of cytosolic redox state in glucose-induced insulin release (GIIR) remains undetermined. The role of cytosolic redox state has been assessed (as reflected by the lactate/pyruvate ratio) in nutrient- and non-nutrient-induced insulin release using a recently established glucose-sensitive clonal β-cell line (BRIN-BD11). Long-term exposure to the NAD + precursor vitamin nicotinic acid (NA, 100 μM) was used to promote a more oxidized state in the cytosol. Glucose (2–16 mM) evoked a dose-dependent rise in the cytosolic NADH/NAD + ratio which was linearly related to the extent of GIIR. NA suppressed the glucose-induced rise in the NADH/NAD + ratio and concomitantly reduced GIIR by 44%. It also inhibited, by 47%, the average glucose-induced rise in cytosolic free Ca 2+ concentration ([Ca 2+] i , assessed by fura-2 microfluorometry from single cells). The latter effect was not accounted for by a reduction in the activity of voltage-sensitive Ca 2+ channels, inasmuch as both high K +- and tolbutamide-induced [Ca 2+] i rises remained insensitive to NA exposure. NA did not affect insulin release evoked by any of the depolarizing agents, indicating that steps in the stimulus-secretion coupling cascade distal to Ca 2+ influx are insensitive to changes in the cytosolic redox state. It is concluded that GIIR is partially controlled by the cytosolic redox state. Moreover, the impairment in GIIR, caused by a shift toward a more oxidized state in the cytosol, originates from an attenuated [Ca 2+] i response. The latter is likely mediated by the influence of cytosolic redox state on specific metabolic pathways (NADH shuttle systems and/or the malonyl-CoA pathway), leading ultimately to enhancement of the activity of ATP-sensitive K + channels.

Glucose Regulation of Insulin Secretion Independent of the Opening or Closure of Adenosine Triphosphate-Sensitive K+ Channels in Cells

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the β-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on[ Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mm K+, i.e. without pharmacological action on K+-ATP channels,[ Ca2+]i and insulin secretion were already elevated at 3 mm glucose. High glucose (20 mm) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence[ Ca2+]i and insulin secretion at 3 mm glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and β-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between[ Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells.

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo‐3 and CD41

Background: Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Methods: Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. Results: In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Conclusions: Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy. Cytometry 35:302–310, 1999. © 1999 Wiley-Liss, Inc.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.

Early and delayed tolerance to simulated ischemia in heat-preconditioned endothelial cells: a role for HSP27.

An ischemia-mimicking metabolic stress in cultured endothelial cells from the human aorta or umbilical vein caused ATP depletion, a rise in cytosolic free Ca2+, fragmentation and aggregation of actin microfilaments, retraction of the cytoplasm, and disintegration of cell monolayer. Simultaneously, the constitutive heat shock protein 27 (HSP27) underwent dephosphorylation and formed granules inside cell nuclei. Prior heat shock (45 degreesC, 10 min) in confluent cultures conferred two phases (early and delayed) of tolerance to simulated ischemia. Although heat preconditioning did not retard the ATP drop and the free Ca2+ overload within ischemia-stressed cells, each phase of the tolerance was manifested in longer preservation of normal cell morphology during the stress. Cells exhibiting the early tolerance within 3 h after heating altered the F-actin response to ischemic stress; no microfilament debris but, instead, translocation of F-actin to the tight submembranous layer was observed. In contrast, the delayed cytoprotection preserved the preexisting F-actin bundles under simulated ischemia; this happened only after 12- to 14-h post-heat shock recovery, elevating the intracellular HSP content, and was sensitive to blockers of HSP synthesis, cycloheximide and quercetin. The dephosphorylation and intranuclear granulation of HSP27 were markedly suppressed in both phases of the heat-induced tolerance. Without heat pretreatment, similar attenuation of the HSP27 dephosphorylation/granulation and the actin cytoskeleton stability during simulated ischemia were achieved by treating cells with the protein phosphatase inhibitors cantharidin or sodium orthovanadate. We suggest that prior heat shock ameliorates the F-actin response to ischemic stress by suppressing the HSP27 dephosphorylation/granulation; this prolongs a sojourn in the cytosol of phosphorylated HSP27, which protects microfilaments from the disruption and aggregation.

Inhibition of voltage-sensitive calcium channels by the A2A adenosine receptor in PC12 cells.

The role of the A2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca2+ influx induced by membrane depolarization with 70 mM K+ could be inhibited with CGS21680, an A2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp-adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca2+ concentration induced by 70 mM K+, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca2+ influx without VSCC activity after cobalt or 70 mM K+ pretreatment. These data suggest that the A2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A2A receptors induce inhibition of VSCCs as well as ATP-induced Ca2+ influx, the two inhibitory effects are clearly distinct from each other.

Release of 'caged' cytosolic Ca2+ triggers rapid spreading of human neutrophils adherent via integrin engagement.

The role of the transient rise in cytosolic free Ca2+ which occurs during neutrophil adhesion and cell spreading is unclear. In order to establish whether such a Ca2+ signal triggers neutrophil shape change, neutrophils co-loaded with fluo3 and Nitr5 ('caged' Ca2+) were used with rapid-time confocal laser scanning microscopy. Here we show that the photolytic generation of a Ca2+ rise in neutrophils which were adherent to an integrin-engaging surface, triggered a rapid change in cell morphology, with increases in cell diameter of approximately 175% occurring within 90 seconds of the Ca2+ signal. In non-adhered neutrophils or neutrophils on plain glass, no acceleration of the rate of spreading occurring in response to the release of 'caged Ca2+' could be demonstrated. It was concluded that although a rise in cytosolic free Ca2+ was not the sole trigger for neutrophil shape change, with other signals generated by integrin engagement, a rise in cytosolic free Ca2+ accelerated the rate of neutrophil spreading.

A role for local calcium gradients upon hypoxic injury in human umbilical vein endothelial cells (HUVEC)

Upon hypoxic injury, bleb formation is an early event of cell damage observed in a variety of cell types. Although a rise in cytosolic free Ca 2+ ([Ca 2+] i) has been considered to be involved in this process, the exact relationship between these phenomena remains ill-defined. In order to examine the relationship between bleb formation, and [Ca 2+] i or nuclear free Ca 2+ ([Ca 2+] n), we analyzed [Ca 2+] i and [Ca 2+] i in HUVEC during hypoxic injury using confocal laser scanning microscopy. [Ca 2+], and [Ca 2+]n were measured using Fluo-3, and cell viability and mitochondrial membrane potential were assessed by the exclusion of propidium iodide (PI) and rhodamine 123, respectively. After the initiation of hypoxia, [Ca 2+] i and [Ca 2+]n rose gradually up to 15 min reaching peak values of 447 ± 62 and 516 ± 105 nM, respectively, which was accompanied by a decrease in rhodamine 123 fluorescence and an increase in PI-stained cells. Bleb formation was observed after [Ca 2+] i and [Ca 2+] n had reached their peak values and the number of blebs increased thereafter. Confocal sectioning images revealed a localized increase in [Ca 2+] i at the bleb forming site and this localized elevation in [Ca 2+] i was observed before bleb formation in the corresponding area. In conclusion, bleb formation induced by hypoxic stress appears to involve Ca 2+-dependent reactions that are linked to a regional elevation of [Ca 2+] i.

Bcl-2 protects against apoptosis in neuronal cell line caused by thapsigargin-induced depletion of intracellular calcium stores.

The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca2+-ATPase, was investigated in GT1-7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca2+ ([Ca2+]i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca2+ diminished somewhat the size of the initial increase of [Ca2+]i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La3+ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 microM BAPTA/AM, a cytosolic Ca2+ chelator, inhibited the peak [Ca2+]i caused by thapsigargin but did not inhibit the sustained elevation of [Ca2+]i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation ("laddering"), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the protooncogene bcl-2 in GT1-7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl-2 did not alter either basal [Ca2+]i or the elevation of [Ca2+]i induced by thapsigargin. Our results suggest that abnormal Ca2+ release from endoplasmic reticulum caused by thapsigargin induces GT1-7 death by apoptosis and that this effect does not depend on Ca2+ influx from the extracellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca2+ release in GT1-7 neuronal cells.

Dipeptidyl peptidase IV (CD26) is a receptor for streptokinase on rheumatoid synovial fibroblasts

Plasminogen (Pg) binds to synovial fibroblasts from patients with rheumatoid arthritis with a very high affinity and in a dose-dependent manner. The Pg receptor in these cells is composed of a glycoprotein IIb/IIIa related protein (α IIb β 3 ) in association with dipeptidyl peptidase IV (DPP IV). Pg binds via its kringles to the β 3 subunit of α IIb β 3 , which is in the vicinity of receptor-bound urinary-type plasminogen activator (uPA). Activation of Pg to plasmin (Pm) by uPA induces a conformational change in the molecule, which facilitates the interaction of Pm-carbohydrate chains with DPP IV. This interaction induces a rise in cytosolic free Ca 2+ concentration. The streptococcal Pg activator streptokinase (SK) also binds to rheumatoid synovial fibroblasts with high affinity and in a dose-dependent manner. By contrast, normal synovial fibroblasts do not bind SK. Cell-bound SK can activate Pg and induces a cytosolic Ca 2+ mobilization which differs in nature from the response observed after activation by uPA. SK binding can be inhibited by fibronectin (FN), but not by Pg- or Pm-derived heavy chain. The receptor for SK was purified by affinity chromatography and identified as DPP IV. SK and FN bind to DPP IV via the amino acid sequence LTSRPA, common to both ligands. Our studies suggest that Pg/Pm binding to rheumatoid synovial fibroblasts can be regulated via two distinct types of binding sites on DPP IV. The first site involves direct binding via a lectin-like interaction. The second site functions in association with FN/SK. These findings suggest an important role of DPP IV in the regulation of Pg binding and activation on the surface of rheumatoid synovial fibroblasts.

Imaging bioluminescent indicators shows Ca2+ and ATP permeability thresholds in live cells attacked by complement.

A series of permeability thresholds to Ca2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase-aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 micrograms/ml no loss of luciferase-aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted.

Signal transduction mechanisms in nutrient-induced insulin secretion.

The knowledge of the mechanism whereby glucose and other fuel stimuli promote the release of insulin by the pancreatic beta cell remains fragmentary. The closure of metabolically sensitive K+ channels and a rise in cytosolic free Ca2+ are key features of beta-cell metabolic signal transduction. However, these two signalling events do not account for the dose dependence of glucose-induced insulin secretion. In fact, recent evidence indicates that there are KATP channel and Ca2+ independent pathway(s) of beta-cell activation which remain to be defined. In this review, we have limited our attention to the recent developments in our understanding of the mode of action of nutrient secretagogues. A particular emphasis is placed in summarising the evidence in support of two new concepts: 1) oscillations in the glycolytic pathway and beta-cell metabolism contribute to the oscillatory nature of beta-cell ionic events and insulin secretion; 2) malonyl-CoA and long chain acyl-CoA esters may act as metabolic coupling factors in beta-cell signalling. Finally, we propose that the altered expression of genes encoding enzymes in the pathway of malonyl-CoA formation and fatty acid oxidation contributes to the beta-cell insensitivity to glucose in some patients with non-insulin-dependent diabetes mellitus.

A2A adenosine receptors inhibit ATP-induced Ca2+ influx in PC12 cells by involving protein kinase A.

The regulatory role of A2A adenosine receptors in P2 purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca2+ concentration ([Ca2+]i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca2+ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca2+]i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp-adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A2A adenosine receptors leads to inhibition of P2 purinoceptor-mediated Ca2+ influx through ATP-gated cation channels and involves protein kinase A.

Effect of ethanol on adenosine triphosphate, cytosolic free calcium, and cell injury in rat hepatocytes. Time course and effect of nutritional status.

The events implicated in the early phases of acute ethanol-induced hepatocyte injury and their relation with the nutritional status of the liver are not clearly defined. We aimed to determine the effect of ethanol on ATP and cytosolic free Ca2+ in hepatocytes isolated from fed or fasted rats. Cell injury was assessed by LDH release and trypan blue uptake, ATP by [31P]NMR spectroscopy, and cytosolic free Ca2+ with aequorin. In control conditions, cells from fasted animals had a lower ATP level (-52%) and a higher cytosolic free Ca2+ (+101%) than did those isolated from fed animals. Ethanol caused a dose-dependent cell injury in both groups. At all ethanol doses, greater, damage occurred when using hepatocytes isolated from fasted rats. In both groups, a dose-dependent decrease in ATP content and a rise in cytosolic free Ca2+ were seen. The magnitude of these changes were significantly greater in the fasted group. In conclusion, these data showed that fasting affects the energy status and cytosolic free calcium level in hepatocytes; ethanol causes a dose-dependent cell injury that occurs in association with a fall in ATP and a rise in cytosolic free Ca2+ levels. The nutritional status of an animals is an important determinant of the severity of ethanol-induced damage to liver cells.

Growth-promoting effects of Ca2+-mobilizing agents in hepatocytes: Lack of correlation between the acute activation of phosphoinositide-specific phospholipase C and the stimulation of DNA synthesis by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2α

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide-specific phospholipase C (PI-PLC) and mobilize Ca2+, the role of PI-PLC in the growth-stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the beta-adrenoceptor blocker timolol) or PGF2 alpha, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5-trisphosphate (InsP3) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI-PLC, measured as the early rise (peak 15-60 s) in InsP3, was 8-10-fold with vasopressin or angiotensin II, 3-4-fold with norepinephrine, and approximately 2-fold with PGF2 alpha. For all the agonists, a rise in cytosolic free Ca2+ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP3. However, the growth-stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI-PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine approximately PGF2 alpha > angiotensin II > vasopressin. Also, norepinephrine, PGF2 alpha, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP3. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP3 responses were not. The results show that the extent of the initial activation of PI-PLC is not the determinant for the magnitude of the growth effects of Ca(2+)-mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI-PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI-PLC-independent, mechanisms are required.

Modulation of the hepatic α1‐adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca2+‐dependent and independent responses

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.