The Riddle of Formycin A Insulinotropic Action

Formycin A augments insulin release evoked by glucose (5.6 mmor more), this effect not being rapidly reversible. The mechanism responsible for the insulinotropic action of formycin A was investigated in isolated pancreatic islets. It could not be ascribed to facilitation of glucose metabolism. On the contrary, formycin A inhibited glucose oxidation, lowered ATP content, and impaired glucose-stimulated protein biosynthesis. The insulinotropic action of formycin A was apparently attributable to its conversion to formycin A 5′-triphosphate, both this process and the secretory response to formycin A being abolished by the inhibitor of adenosine kinase 5-iodotubercidin. In agreement with the latter view, adenosine receptor antagonists such as 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine failed to suppress and, instead, augmented the insulinotropic action of formycin A. Unexpectedly, however, formycin A failed to decrease⁸⁶Rb efflux, this coinciding with a low efficiency of formycin A 5′-triphosphate to inhibit K_ATP-channel activity in excised membranes and with the fact that formycin A increased gliben-clamide-stimulated insulin release. The secretory response to formycin A represented a Ca²⁺-dependent process suppressed in the absence of extracellular Ca²⁺or presence of verapamil and associated with an increased net uptake of⁴⁵Ca. Nevertheless, the view that formycin A exerts any major effect upon intracellular Ca²⁺redistribution, protein kinase C activity, or cyclic AMP net production also met with objections such as the minor secretory effect of formycin A in islets exposed to a high concentration of K⁺in the presence of a diazoxide analog, the resistance of formycin A insulinotropic action to bisindolylmaleimide, the poor increase of cyclic AMP content in formycin A-stimulated islets, and the pronounced enhancement by forskolin or theophylline of insulin release from islets exposed to formycin A. It is concluded, therefore, that the mechanism of action of formycin A in the pancreatic β-cell remains to be elucidated.     

Calcium antagonists and islet function: VII. Effect of calcium deprivation

Summary The role of extracellular Ca²⁺ in the regulation of islet function is investigated. Decreasing extracellular Ca²⁺ concentrations cause a dose-related inhibition of glucose-induced insulin release. Whereas the efflux of⁴⁵Ca from perifused islets is transiently increased on exposure to Ca²⁺-deprived media, it is unaffected by a partial lowering of the extracellular Ca²⁺ concentration. Under the latter condition, therefore, the observed reduction in the size of the islets' exchangeable calcium pool(s) appears to be due to reduced Ca²⁺ entry. The proper effect of glucose on Ca handling by the islets is apparently not affected by a lowering in the extracellular Ca²⁺ concentration. Nevertheless, in islets exposed to glucose and incubated in Ca²⁺-deprived media, glucose uptake and oxidation and lactate output are decreased, whereas the islet ATP level is increased, as if extracellular Ca²⁺ shortage were to affect not only the cellular pool of Ca regulating insulin release, but also energy-consuming processes possibly located at the cell membrane.     

The stimulus-secretion coupling of glucose-induced insulin release: effects of valinomycin upon pancreatic islet function.

In isolated rat pancreatic islets, valinomycin (0.01 to 1.0 μm) caused a dose-related facilitation of ⁸⁶Rb⁺ outflow and a dose-related inhibition of the glucose-induced changes in both outflow and net uptake of ⁸⁶Rb⁺. At high concentrations (0.1–1.0 μm), the ionophore also inhibited the oxidation of glucose and endogenous nutrients, decreased the adenylate charge, and lowered the concentration of reduced pyridine nucleotides in the islet cells. However, as little at 1.0 to 10.0 nm valinomycin caused anomalies in the handling of ⁴⁵Ca²⁺ (suppression of the early inhibitory effect of glucose upon ⁴⁵Ca²⁺ efflux, and reduction in the amount of ⁴⁵Ca²⁺ recovered in the islets after an extensive washing procedure) and inhibition of insulin release. Moreover, when the effect of glucose upon K⁺ conductance was abolished by high concentrations of valinomycin (0.1–1.0 μm), the glucose-induced secondary rise in ⁴⁵Ca²⁺ efflux was still observed. These findings suggest that the effects of glucose upon ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ handling, respectively, although normally concomitant with one another, can be dissociated, in part at least, from one another. It is concluded that the glucose-induced reduction in K⁺ outflow may be unnecessary for the sugar to cause a partial remodeling of Ca²⁺ fluxes in the islet cells.     

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO₂, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ? l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K⁺, Ca²⁺) across membrane systems and, hence, regulates the net uptake of Ca²⁺ and subsequent release of insulin. The effect of l-lactate upon Ca²⁺ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.     

The effects of d- and l-glyceraldehyde on glucose oxidation, insulin secretion and insulin biosynthesis by pancreatic islets of the rat

d-glyceraldehyde stimulated insulin secretion from isolated rat pancreatic islets in static incubation and perifusion systems. At low concentrations (2–4 mM) d-glyceraldehyde was a more potent secretagogue than glucose. The insulinotropic action of 15 mM d-glyceraldehyde was not affected by d-mannoheptulose, was potentiated by cytochalasin B (5 μg/ml) and theophylline (4 mM), and was inhibited by both adrenalin (2 μM) and somatostatin (10 μg/ml). D-glyceraldehyde at a concentration of 1.5 mM produced a 10-fold increase of l-[4,5-³ H]leucine incorporation into proinsulin and insulin without a significant increase into other islet proteins. Glucose at 1.5 mM did not stimulate proinsulin biosynthesis. d-Glyceraldehyde at concentrations higher than 1.5 mM, in marked contrast to glucose, progressively inhibited incorporation of labelled leucine into proinsulin + insulin and other islet proteins. d-glyceraldehyde also inhibited the oxidation of glucose. l-Glyceraldehyde did not stimulate proinsulin biosynthesis and had less effect than the d-isomer on insulin release and glucose oxidation. The results strongly suggest that metabolites below d-glyceraldehyde-3-P are signals for insulin biosynthesisand release. Interaction of d-glyceraldehyde with a “membrane receptor” cannot, however, be excluded with certainty.     

Effects of noradrenaline on45Ca2+ efflux from isolated rat islets of Langerhans

Noradrenaline caused a prompt but transient increase in the rate of⁴⁵Ca²⁺ efflux from isolated rat islets of Langerhans perifused in Ca²⁺ depleted medium. The response was modest in size and was unaffected by isosmotic replacement of NaCl with choline chloride or by inclusion of 0.5 mM dibutyryl cAMP in the perifusion medium, suggesting that it was not mediated by Na+: Ca²⁺ exchange nor by lowered cAMP. Despite its effect on⁴⁵Ca²⁺ efflux, noradrenaline treatment did not alter the kinetics of⁴⁵Ca²⁺ efflux in response to the muscarinic agonist, carbamylcholine, nor did it change the magnitude of the response to this agent. Simultaneous introduction of 20 mM glucose with noradrenaline prevented a rise in⁴⁵Ca²⁺ efflux and indeed resulted in inhibition of⁴⁵Ca²⁺ efflux. The data suggest that noradrenaline does not directly activate the mechanisms which regulate Ca²⁺ extrusion from islets cells, and they do not support a primary role for the Ca²⁺ efflux response in mediating adrenergic inhibition of insulin secretion.     

Effects on N6, O2′-dibutyryladenosine-3′,5′ cyclic monophosphate on calcium accumulation by mouse mastocytoma cells

The accumulation of ⁴⁵Ca²⁺ by intact mouse mastocytoma cells was examined before and after treatment of the cells with N⁶,O^2′-dibutyryladenosine 3′,5′, cyclic monophosphate and theophylline to inhibit growth. In the presence of phosphate either glycolysis, respiration or ATP supported ⁴⁵Ca²⁺ uptake by the cells and in each case the accumulated ⁴⁵Ca²⁺ appeared to be retained by mitochondria. Inhibition of growth by drug treatment for 20h increased subsequent ⁴⁵Ca²⁺ accumulation when cells were incubated with ⁴⁵CaCl₂, succinate and phosphate. Since prior drug treatment did not increase ⁴⁵Ca²⁺ accumulation with glucose, ATP or malate the drugs appeared to increase ⁴⁵Ca²⁺ accumulation by affecting succinate metabolism.     

Glucose-stimulated Single Pancreatic Islets Sustain Increased Cytosolic ATP Levels during Initial Ca2+ Influx and Subsequent Ca2+ Oscillations

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca²⁺ levels throughout the islets during high glucose conditions. This Ca²⁺ elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K⁺ channels, leading to the depolarization of membranes, which in turn induces Ca²⁺ elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca²⁺ levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca²⁺ dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca²⁺ levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca²⁺ increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca²⁺ influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca²⁺ level in the subsequent oscillation phases. Importantly, Ca²⁺ oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion.     

Testosterone rapidly stimulates insulin release from isolated pancreatic islets through a non-genomic dependent mechanism.

The action of testosterone on the 45Ca2+ uptake and insulin secretion was studied in short-term experiments using isolated pancreatic islets of Langerhans. Testosterone (1 microM) stimulated 45Ca2+ uptake within 60 seconds of incubation on similar proportion than tolbutamide. Also, the hormone rapidly increased insulin release (34%; 180 seconds) on the presence of non-stimulatory concentrations of glucose (3 mM). Impermeant testosterone-BSA significantly stimulated the secretion of insulin to a lower percentage (10%). The action of the hormone is specific--neither 17beta-E2 nor progesterone stimulated insulin secretion in the presence of 3 mM glucose. The action of testosterone on insulin secretion was dose-dependent, and at rat plasma physiological concentrations (25 nM), stimulus was 17% (p < 0.05). In conclusion, in isolated pancreatic islets experiments, physiological concentration of testosterone rapidly stimulate insulin secretion and 45Ca2+ uptake through a membrane bound mechanism.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

cAMP-enhancing agents “permit” stimulus-secretion coupling in canine pancreatic islet β-cells

Isolated canine islets of Langerhans differ from isolated islets of other species (including rodents and man) in that elevated glucose concentrations are unable to stimulate insulin secretion. Here we demonstrate that addition to the perifusate of isobutylmethylxanthine (IBMX), forskolin or 8-CPT-cAMP, all of which enhance cytosolic cAMP, permits insulin secretion in response to glucose, leucine or tolbutamide. These cAMP enhancers increase secretogogue-induced electrical activity in β-cells and restore depolarization-induced, Ca²⁺-dependent granule exocytosis measured as stepwise increases in membrane capacitance. We propose that the primary permissive action of cAMP is to tightly link Ca²⁺ entry to insulin granule release, while a secondary action is to tighten the link between glucose metabolism and cell depolarization.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Effect of estradiol on Ca<Superscript>2+</Superscript> release from intracellular stores in porcine oocytes stimulated by prolactin,theophylline, or guanosine triphosphate

The interaction between prolactin and theophylline as well as between prolactin and guanosine triphosphate during Ca²⁺ release from intracellular stores of estradiol-treated porcine oocytes isolated from the ovary at the stage of follicular growth were studied using fluorescent Ca²⁺-sensitive probe chlortetracycline. In the absence of estradiol, prolactin or theophylline induced Ca²⁺ release from intracellular stores; however, no increase in Ca²⁺ release was observed after their combined action. Conversely, Ca²⁺ release from intracellular stores increased only after the combined exposure to prolactin and theophylline in the presence of estradiol. In the absence of estradiol, guanosine triphosphate induced calcium release alone and together with prolactin. Protein kinase C regulated Ca²⁺ release from intracellular stores after the combined exposure to prolactin and theophylline only in the presence of estradiol; while the activation of protein kinase C required no estradiol during the combined exposure to prolactin and guanosine triphosphate. The data obtained indicate the effect of estradiol on Ca²⁺ release from intracellular stores after the combined exposure to prolactin and theophylline, while no such effect was observed after the combined exposure to prolactin and guanosine triphosphate.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca2+ uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of ⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

Mechanism of aluminum inhibition of net ca uptake by amaranthus protoplasts

Calcium ions serve as a second messenger in signal transduction and metabolic regulation. Effects of Al on calcium homeostasis remain to be elucidated. Short-term net ⁴⁵Ca²⁺ uptake by Amaranthus tricolor protoplasts was monitored from uptake media prepared to test the influence of pH, Al, and various inhibitors. Accumulation of ⁴⁵Ca²⁺ increased during the first 3 to 6 minutes and then leveled off or declined. Al and Ca²⁺ channel blockers (verapamil and bepridil) decreased net ⁴⁵Ca²⁺ uptake. This decrease was more pronounced when Al and bepridil were both present in uptake media, but Al did not aggravate verapamil-induced reduction of net ⁴⁵Ca²⁺ uptake. Erythrosin B and calmidazolium each increased net ⁴⁵Ca²⁺ uptake, probably by interfering with Ca²⁺ efflux. This effect was undetectable in the presence of Al. Mycophenolic acid decreased net ⁴⁵Ca²⁺ uptake; guanosine alleviated this effect. Al-induced reduction of net ⁴⁵Ca²⁺ uptake was not aggravated by mycophenolic acid. Net ⁴⁵Ca²⁺ uptake was generally less at pH 4.5 than at 5.5 for all treatments. It is concluded that Al ions affect net ⁴⁵Ca²⁺ uptake by binding to the verapamil-specific channel site that is different from the bepridil-specific one, as well as by interfering with the action of guanosine 5′-triphosphate-binding proteins.     

Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes.

⁴⁵Ca²⁺ uptake was detected within minutes following addition of T- and B-cell² mitogens to mouse lymphocytes. The T-cell mitogens (Con A and PHA) gave an ~twofold increase in ⁴⁵Ca²⁺ uptake (representing an influx of ~ 130 amol per lymphocyte, corresponding to an increase in average cellular Ca²⁺ of ~0.95 mM). B-cell mitogens which gave the largest ⁴⁵Ca²⁺ uptake (~twofold) were purified LPS preparations from Salmonella minnesota R595 and Escherichia coli 0111:2125. The ⁴⁵Ca²⁺ uptake by rabbit splenocytes using specific anti-b4 allotype antiserum was comparable to that obtained with the two purified LPS preparations. A23187, in low nontoxic doses, gave an ~sixfold increase in ⁴⁵Ca²⁺ uptake with mouse T cells. The ⁴⁵Ca²⁺ uptake was modulated by cyclic nucleotides showing a “yin-yang” effect. The results suggest a possible entry of ⁴⁵Ca²⁺ from the extracellular medium through “gated Ca²⁺ channels” in the plasma membrane into the cytosol by passive diffusion. The Ca²⁺ may be sequestered in the mitochondria, and the excess Ca²⁺ is later effluxed into the extracellular medium. The fact that ⁴⁵Ca²⁺ uptake appears to be one of the earliest events occurring after ligand binding to the cell, together with the demonstration of a Ca²⁺-dependent glucose uptake and a requirement for extracellular Ca²⁺ for DNA synthesis, suggest that, as it is now known to function in many other cellular responses, Ca²⁺ may operate as a second messenger for lymphocyte activation.     

Magnetic field effects on calcium efflux and insulin secretion in isolated rabbit islets of Langerhans

Rabbit islets of Langerhans were exposed at 37 °C for 18 h to a low-frequency-pulsed magnetic field, generated in paired Helmholtz coils. Exposed islets showed a reduction of 26.1 ± 4.3% in ⁴⁵Ca²⁺ content (P < .004). a reduction of 25.1 ± 6.3% in ⁴⁵Ca²⁺ efflux (P < .006), and a reduction of 35.0 ± 8.7% (P < .002) in insulin released during glucose stimulation when compared with appropriate controls.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)     

Comparison of 45Ca2+ uptake activity by microsomes from control and stimulated mouse pancreatic acini

The ability of microsomal preparations to transport ⁴⁵Ca²⁺ was studied in preparations of control and secretagogue-stimulated pancreatic acini. ATP-dependent ⁴⁵Ca²⁺ uptake activity was present in the pancreatic post-mitochondrial supernatant and microsomes but little activity was present in the postmicrosomal supernatant. Treatment of acini with the secretagogues cholecystokinin (CCK) and carbamylcholine (CCh) prior to cell fractionation increased the subsequently measured microsomal ⁴⁵Ca²⁺ uptake. The effect of CCK was maximal after 10 min stimulation and at a not. The effect of CCK was maximal after 10 min stimulation and at a concentration of 1 nM; these conditions are comparable to the effects of CCK on ⁴⁵Ca²⁺ fluxes in intact acini. The increased microsomal ⁴⁵Ca²⁺ uptake induced by CCK was due to an increase in the maximal rate of ⁴⁵Ca²⁺ uptake as there was no effect on the K_m for Ca²⁺ (1 μM). It is concluded that secretagogues increase the ATP-dependent uptake of ⁴⁵Ca²⁺ by an isolated pancreatic microsomal component under the same conditions that also stimulate both digestive enzyme secretion and bi-directional Ca²⁺ movements.