Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINm5F) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101 +/- 5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7 +/- 0.8 min. In contrast to the action of K+, exposure of the RINm5F cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity. This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in beta-cells. Unlike the case in normal beta-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINm5F cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINm5F cells exposed to glucose.     

Accumulation of cadmium in pancreatic beta cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd2+ and the effects of this ion on secretory activity and metabolism were investigated in beta cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 mumol/kg dry wt. After 60 min of incubation in a Ca2+-deficient medium containing 2.5 microM Cd2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca2+. The incorporation of Cd2+ was stimulated either by raising the concentration of glucose to 20 mM or K+ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K+. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd2+ efflux into a Ca2+-deficient medium. Concentrations of Cd2+ up to 2.5 microM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd2+ concentration was above 10 microM. Basal insulin release was stimulated by 5 microM Cd2+. At a concentration of 160 microM, Cd2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the beta cell uptake of Cd2+ is facilitated by the activation of voltage-dependent Ca2+ channels. Apparently, the accumulation of Cd2+ mimics that of Ca2+ also involving a component of intracellular sequestration promoted by glucose.     

Glucose metabolism and glutamate analog acutely alkalinize pH of insulin secretory vesicles of pancreatic beta-cells

We studied acute changes of secretory vesicle pH in pancreatic beta-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 +/- 0.10% at 149 +/- 16 s with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by approximately 0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+ influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethyl ester (GME), a plasma membrane-permeable analog of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM without changing cellular ATP content or cytosolic Ca2+ concentration ([Ca2+]). Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 +/- 0.19% at 38 +/- 2 s. These results indicated that the acutely alkalinizing effect of glucose on beta-cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may have potential relevance to the alteration of secretory function by glutamate.     

Interactions between insulin and alpha 1-adrenergic agents in the regulation of glycogen metabolism in isolated hepatocytes

Addition of insulin to liver cells from fed rats incubated in the absence of other hormones resulted in a 2-fold increase in glycogen synthase activity. This direct effect of insulin has been characterized and compared with the antagonism by insulin of alpha 1-adrenergic effects on glycogen metabolism. The activation of glycogen synthase by insulin developed slowly (20-25 min) and was most effective when the enzyme was partially preactivated by glucose. With glucose concentrations above 15 mM the effects of insulin and glucose were additive. In contrast to glucose, which caused inverse changes in phosphorylase and glycogen synthase activity, insulin activated glycogen synthase without affecting phosphorylase a. Treatment of hepatocytes with phenylephrine led to an activation of phosphorylase and inactivation of glycogen synthase, which could be partially blocked by insulin. This antagonistic effect of insulin was rapid (complete within 5 min of insulin addition) and showed an identical time course for both enzymes. The activation of glycogen synthase by insulin and inactivation by phenylephrine both resulted principally from alterations in the Vmax. Insulin added alone did not alter the basal cytosolic free Ca2+ concentration, which was 160 nM as measured with Quin 2 as an intracellular Ca2+ indicator. Both the magnitude and the initial rate of cytosolic free Ca2+ increase induced by phenylephrine were reduced by about 50% in cells pretreated with insulin. It is concluded that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase, whereas insulin antagonizes the effects of alpha 1-agonists by interfering with their ability to elevate cytosolic free Ca2+.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

Glucose increases cytosolic calcium concentration and inositol lipid metabolism in HIT-T15 cells

We have investigated the effects of glucose on cytosolic free calcium concentration in the insulin-secreting cell line HIT-T15. Addition of glucose (10 mM) caused a 20-75% increase in cytosolic [Ca2+] within 5 minutes compared to controls in the absence of glucose. A maximal increase in cytosolic [Ca2+] was obtained with 5 mM glucose. The magnitude of the response was markedly dependent upon the concentration of extracellular Ca2+, and the rise in cytosolic [Ca2+] was inhibited by verapamil. Cytosolic [Ca2+] was greatly increased by depolarization of the cells with KCl (50 mM), whereas carbamylcholine had no apparent effect. Glucose and KCl were also effective in stimulating insulin release from HIT cells, although carbamylcholine was again ineffective. The secretory response to glucose was also found to be directly related to the concentration of extracellular [Ca2+]. Glucose and KCl, but not carbamylcholine, were found to slightly enhance the production of [3H]-inositol trisphosphate in HIT cells pre-labelled with myo-[3H]-inositol, indicating a modest stimulation of inositol lipid hydrolysis.     

P2Y-purinoceptor mediated inhibition of L-type Ca2+ channels in rat pancreatic beta-cells

We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway.     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.     

Calcium-dependent effect of gentamicin on glucose formation in isolated rabbit kidney-cortex tubules.

In contrast to the inhibition by gentamicin of glucose production from propionate, pyruvate and lactate in renal tubules incubated at 2.5 mM Ca2+, this antibiotic does not affect gluconeogenesis from propionate and lactate, and significantly stimulates this process from other substrates at 0.5 mM Ca2+. This may be due to the gentamicin-induced increase of the cytosolic manganese content (from 1.7 to 2.7 nmol/mg protein), resulting in a stimulation of cytosolic phosphoenolpyruvate carboxykinase activity. At 2.5 mM Ca2+ the cytosolic Mn content (2.7 nmol/mg protein) seems to be high enough to accomplish activation of the enzyme.     

Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract

We show in the accompanying paper that the steady-state level of free Ca2+ maintained by the organelles of permeabilized RINm5F insulinoma cells varies inversely with the ATP/ADP ratio when this ratio is set by addition of creatine phosphokinase and fixed ratios of creatine to creatine phosphate. We, therefore, asked whether acute cyclic alterations in the cytosolic ATP/ADP ratio in the range known to modulate O2 consumption might be involved in regulating the physiological activity of Ca2+ -ATPases and the cytosolic free Ca2+ level. To explore this hypothesis we combined two experimental systems: 1) permeabilized RINm5F insulinoma cells that can maintain a low medium Ca2+ concentration and 2) a cell-free extract of rat skeletal muscle that spontaneously exhibits oscillatory behavior of glycolysis and linked oscillations in the ATP/ADP ratio, when provided with glucose. The free Ca2+ level maintained by the permeabilized cells oscillated in phase with the glycolytic oscillations and correlated closely with the ATP/ADP ratio but not with glucose 6-phosphate, fructose 6-phosphate, orthophosphate, or pH. When glucokinase replaced hexokinase as the glucose phosphorylating enzyme, Ca2+ oscillations were induced by increasing the glucose concentration from 2 to 8 mM. The results demonstrate a link between metabolite changes and free Ca2+ levels in a reconstituted physiological system. They support a model in which oscillations in glycolysis and the ATP/ADP ratio may cause oscillations in cytosolic free Ca2+, beta-cell electrical activity, and insulin release.     

Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.     

Role of voltage-dependent Na+ channels for rhythmic Ca2+ signalling in glucose-stimulated mouse pancreatic beta-cells.

The putative role of voltage-dependent Na+ channels for glucose induction of rhythmic Ca2+ signalling was studied in mouse pancreatic beta-cells with the use of the Ca2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na+ channel blocker tetrodotoxin. During exposure to 1-10 microM veratridine, which facilitates the opening of voltage-dependent Na+ channels, the slow oscillations were replaced by repetitive and pronounced [Ca2+]i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca2+]i transients were critically dependent on the influx of Ca2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca2+]i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na+ channels is a useful approach for amplifying Ca2+ signals for insulin release.     

Membrane potential dependent modulations of calcium oscillations in insulin-secreting INS-1 cells

This study was undertaken to examine the role of K(+) channels on cytosolic Ca(2+) ([Ca(2+)](i)) in insulin secreting cells. [Ca(2+)](i) was measured in single glucose-responsive INS-1 cells using the fluorescent Ca(2+) indicator Fura-2. Glucose, tolbutamide and forskolin elevated [Ca(2+)](i) and induced [Ca(2+)] oscillations. Whereas the glucose effect was delayed and observed in 60% and 93% of the cells, in a poorly and a highly glucose-responsive INS-1 cell clone, respectively, tolbutamide and forskolin increased [Ca(2+)](i) in all cells tested. In the latter clone, glucose induced [Ca(2+)](i) oscillations in 77% of the cells. In 16% of the cells a sustained rise of [Ca(2+)](i) was observed. The increase in [Ca(2+)](i) was reversed by verapamil, an L-type Ca(2+) channel inhibitor. Adrenaline decreased [Ca(2+)](i) in oscillating cells in the presence of low glucose and in cells stimulated by glucose alone or in combination with tolbutamide and forskolin. Adrenaline did not lower [Ca(2+)](i) in the presence of 30mM extracellular K(+), indicating that adrenaline does not exert a direct effect on Ca(2+) channels but increases K(+) channel activity. As for primary b-cells, [Ca(2+)](i) oscillations persisted in the presence of closed K(ATP) channels; these also persisted in the presence of thapsigargin, which blocks Ca(2+) uptake into Ca(2+) stores. In contrast, in voltage-clamped cells and in the presence of diazoxide (50mM), which hyperpolarizes the cells by opening K(ATP) channels, [Ca(2+)](i) oscillations were abolished. These results support the hypothesis that [Ca(2+)](i) oscillations depend on functional voltage-dependent Ca(2+) and K(+) channels and are interrupted by a hyperpolarization in insulin-secreting cells.     

Evidence for the presence of active and inactive forms of cytosolic triiodothyronine binding protein in rat kidney: cooperative action of Ca2+ in NADPH activation

The effect of NADPH and Ca2+ on 3, 5, 3'-L-triiodothyronine (T3) binding to cytosolic T3 binding protein (CTBP) in rat kidney was investigated in vitro. Extraction of rat kidney cytosol with 10% charcoal at 4 degrees C for 30 min. inactivated specific T3 binding. The decreased T3 binding activity in extracted cytosol could be restored by adding NADPH or Ca2+ to the incubation medium. Each substance increased the capacity for T3 without changes in the affinity for T3. The T3 binding was maximally increased by 25 microM NADPH or by 1.0 mM Ca2+ in the presence of 0.1 mM EDTA. The increase in T3 binding, induced by 25 microM NADPH, was enhanced by adding 0.2-1.0 mM Ca2+ in a concentration dependent-manner. The increase in T3 binding, induced by 1.0 mM Ca2+, was also enhanced by adding 3.125-25.0 microM NADPH. The NADPH-induced increase in T3 binding capacity was amplified by Ca2+ in a multiplicative manner. The results suggested that Ca2+ cooperatively augmented an NADPH function in cytosolic T3 binding.     

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.     

Mechanisms of coronary artery depolarization by uridine triphosphate

We sought to define the basic mechanisms by which pyrimidine nucleotides constrict rat coronary resistance arteries. Uridine triphosphate (UTP) caused a dose-dependent constriction in coronary arteries stripped of endothelium. UTP also depolarized and increased cytosolic Ca2+ in coronary smooth muscle cells. Nisoldipine, an antagonist of voltage-operated Ca2+ channels, blocked the rise in cytosolic Ca2+ and reduced UTP-induced vasoconstriction by approximately 75% which suggests a prominent role for depolarization in this constrictor response. The ionic basis of UTP-induced depolarization was subsequently explored in coronary smooth muscle cells using whole-cell patch-clamp electrophysiology. In the absence of K+ and with CsCl in the pipette, UTP (40 microM) activated a sustained inwardly rectifying current (-0.66 +/- 0.10 pA/pF at -60 mV). A 100 mM reduction in bath Na+ shifted the reversal potential of this current (from -2 +/- 1 to -28 +/- 4 mV) and reduced the magnitude (from -2.26 +/- 0.61 to -0.51 +/- 0.11 pA/pF). In addition to activating a depolarizing cation current, UTP inhibited hyperpolarizing outward currents. Specifically, UTP inhibited ATP-sensitive and voltage-dependent K+ currents yet had no effect on inwardly rectifying and Ca2+-activated K+ channels. This study indicates that electromechanical coupling is integral to pyrimidine-induced constriction in coronary resistance arteries.     

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.     

Parallel oscillations of intracellular calcium activity and mitochondrial membrane potential in mouse pancreatic B-cells

Insulin secretion in normal B-cells is pulsatile, a consequence of oscillations in the cell membrane potential (MP) and cytosolic calcium activity ([Ca(2+)](c)). We simultaneously monitored glucose-induced changes in [Ca(2+)](c) and in the mitochondrial membrane potential DeltaPsi, as a measure for ATP generation. Increasing the glucose concentration from 0.5 to 15 mM led to the well-known hyperpolarization of DeltaPsi and ATP-dependent lowering of [Ca(2+)](c). However, as soon as [Ca(2+)](c) rose due to the opening of voltage-dependent Ca(2+) channels, DeltaPsi depolarized and thereafter oscillations in [Ca(2+)](c) were parallel to oscillations in DeltaPsi. A depolarization or oscillations of DeltaPsi cannot be evoked by a substimulatory glucose concentration, but Ca(2+) influx provoked by 30 mM KCl was followed by a depolarization of DeltaPsi. The following feedback loop is suggested: Glucose metabolism via mitochondrial ATP production and closure of K(+)(ATP) channels induces an increase in [Ca(2+)](c). The rise in [Ca(2+)](c) in turn decreases ATP synthesis by depolarizing DeltaPsi, thus transiently terminating Ca(2+) influx.     

Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells

Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.