Calcium signalling in glial cells

Calcium signals are the universal way of glial responses to the various types of stimulation. Glial cells express numerous receptors and ion channels linked to the generation of complex cytoplasmic calcium responses. The glial calcium signals are able to propagate within glial cells and to create a spreading intercellular Ca2+ wave which allow information exchange within the glial networks. These propagating Ca2+ waves are primarily mediated by intracellular excitable media formed by intracellular calcium storage organelles. The glial calcium signals could be evoked by neuronal activity and vice versa they may initiate electrical and Ca2+ responses in adjacent neurones. Thus glial calcium signals could integrate glial and neuronal compartments being therefore involved in the information processing in the brain.     

Calcium signalling in glial cells

Various electrical, mechanical, and chemical stimuli, including the influences of neurotrasmitters, neuromodulators, and hormones, trigger complex changes in [Ca²⁺] _i in all types of glial cells. Glial [Ca²⁺] _i responses are controlled by coordinated activity of several molecular cascades. The initiation of [Ca²⁺] _i signal in glial cells results from activation of either plasmalemmal, or intracellular Ca²⁺ permeable channels. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide the means for intracellular and intercellular information coding. Furthermore, glial [Ca²⁺] _i signals can travel with no decrement within glial networks. These intercellular Ca²⁺ waves can be regarded as a substrate for information exchange between the glial cells. Neuronal activity can trigger [Ca²⁺] _i signals in neighboring glial cells and, moreover, there is some evidence that glial [Ca²⁺] _i waves can activate neuronal electrical and/or [Ca²⁺] _i , responses. Glial Ca²⁺ signalling can be regarded as a form of glial excitability.     

Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis

Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes that catalyze the hydrolysis of cAMP and cGMP, thereby restricting the activity of these second messengers in cells. A unique ability to shape gradients of cyclic nucleotides and compartmentalize their signaling implies a high potency and a rapid action of PDEs. However, it has not been demonstrated how fast PDEs can hydrolyze cAMP in a living system. Here we perform a real-time monitoring of PDE2 activity in aldosterone-producing adrenal cells using a recently developed genetically encoded, fluorescent cAMP sensor, which reveals enormously rapid kinetics of cAMP degradation. Activation of PDE2 results in a rapid decrease of intracellular cAMP from high micromolar to the sub-micromolar range within a few seconds. Moreover, the kinetics of atrial natriuretic peptide-stimulated PDE2 activity (measured as decline of cAMP) are much faster than the speed of ACTH and isoprenaline-induced cAMP-synthesis (measured as cAMP accumulation) in the cells, revealing high catalytic activity and fast action of PDEs in regulating cAMP signaling in a physiological system.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

Potentiation of guanosine 3':5'-monophosphate accumulation in C-6 glial tumor cells.

C-6 glial tumor cells treated with norepinephrine and sodium azide accumulated cyclic GMP to concentrations approximately 10-fold greater than the sum of the separate responses. Isoproterenol, but not phenylephrine, was an effective substitute for norepinephrine, and the response was blocked by propranolol and sotalol. Nitroprusside, but neither cyanide nor isobutyl-methylxanthine, replaced azide. The potentiation was not affected by removal of CA2+ OR Na+ from the extracellular medium and was not blocked by cocaine. The potentiative accumulation of cyclic GMP in C-6 cells differs from the recently described stimulation by catecholamines of soluble guanylate cyclase of renal cortex. The potentiative phenomenon is compared with the few known instances in which cyclic AMP augments cyclic GMP formation and may be associated with synergistic modifications of cellular functions.     

Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells.

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP gamma S in permeabilized cells. 2. Nicotinic stimulation, elevated K+, and muscarinic stimulation cause similar production of inositol phosphates (D. A. Eberhard and R. W. Holz, J. Neurochem. 49:1634-1643, 1987). Nicotinic stimulation and elevated K+ but not muscarinic stimulation increased the levels of [3H]inositol-labeled phosphatidylinositol phosphate by 30-60% and [3H]phosphatidylinositol bisphosphate by 25-30%. The increase required Ca2+ in the medium, was maximal by 1-2 min, and was not preceded by an initial decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. 3. In digitonin-permeabilized cells, Ca2+ caused as much as a twofold increase in [3H]phosphatidylinositol phosphate and [3H]phosphatidylinositol bisphosphate. Similarly, Ca2+ enhanced the production of [32P]phosphatidylinositol phosphate and [32P]phosphatidylinositol bisphosphate in the presence of [gamma-32P]ATP. In contrast, GTP gamma S in permeabilized cells decreased polyphosphoinositides in the presence or absence of Ca2+. 4. The ability of Ca2+ to increase the levels of the polyphosphoinositides decayed with time after permeabilization. The effect of Ca2+ was increased when phosphoesterase and phospholipase C activities were inhibited by neomycin. 5. These observations suggest that Ca2+ specifically enhances polyphosphoinositide synthesis at the same time that it activates phospholipase C.     

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.     

FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.     

Alterations of glial tumor cell Ca2+ metabolism and Ca2+-dependent cAMP accumulation by phorbol myristate acetate

C6 glial tumor cells exposed to phorbol myristate acetate (PMA) possessed lowered cAMP content, reduced ability to accumulate cAMP in response to norepinephrine or cholera toxin, and a 3-fold increase in the concentration of norepinephrine producing 50% of the maximal rate of cAMP accumulation. Detectable effects on cAMP accumulation occurred within 10 min of exposure to PMA, and prominent effects by 2 h. PMA similarly affected cells pretreated with cycloheximide. In contrast, Ca2+-depleted preparations of control and PMA-treated cells accumulated cAMP identically in response to norepinephrine or cholera toxin. Ca2+ restoration, which increased the rate of cAMP accumulation in control cells severalfold, did not enhance cAMP accumulation in PMA-treated cells. Neither high catecholamine nor high extracellular Ca2+ concentrations reversed the suppression of cAMP accumulation by PMA. Trifluoperazine, which inhibited the Ca2+-dependent component of norepinephrine-stimulated cAMP accumulation in control cells, did not significantly reduce norepinephrine-stimulated cAMP accumulation in PMA-treated cells. Cell free preparations of control and PMA-treated cultures did not differ significantly in calmodulin content or in Ca2+-stimulated adenylate cyclase, Ca2+-dependent cAMP phosphodiesterase, and (Ca2+-Mg2+)-ATPase activities. The Ca2+ content, however, of intact cells decreased with time of PMA treatment. Within minutes after exposure to PMA, the ability of Ca2+-depleted cells to take up 45Ca was significantly reduced. Both 45Ca uptake and Ca2+-dependent cAMP accumulation were reduced over the same PMA concentration range.     

Calcium dependence of beta-adrenoceptor mediated cyclic AMP accumulation in human lymphocytes

In intact human lymphocytes, cyclic AMP accumulation in response to isoproterenol was inhibited by 5 mM EDTA, by deletion of calcium ions from the medium and by 1 mM lanthanum chloride, but not by 1 microM verapamil or by 10 microM nifedipine. A23187 caused a modest increase in cyclic AMP content. Exposure of lymphocytes to 5 microM 1-isoproterenol desensitized the cells to subsequent beta-adrenergic stimulation, reducing cyclic AMP accumulation. With higher concentrations of 1-isoproterenol (50 microM), receptor density was reduced as well. None of the above agents attenuated losses in agonist-stimulated cyclic AMP accumulation induced by treatment with 5 microM isoproterenol for 90 min. These data suggest that calcium ions, both those present in the extracellular medium and those bound to the plasma membrane, are required for isoproterenol-stimulation of adenylate cyclase. In addition, it appears that neither the presence of extracellular calcium ions nor full activation of adenylate cyclase are required for desensitization.     

Glucosphingolipid dependence of hormone-stimulated inositol trisphosphate formation

The modulatory role of endogenous cellular glycosphingolipids in bradykinin-stimulated myo-inositol 1,4,5-trisphosphate (InsP3) formation by MDCK cells was evaluated utilizing the glucosylceramide synthase inhibitor, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Bradykinin-stimulated InsP3 formation in intact cells and in isolated plasma membranes was significantly enhanced when cells were first depleted of their glucosphingolipids. The effect of glucosphingolipid depletion on phospholipase C activity was dependent on the duration of exposure to the inhibitor and the cellular level of glucosylceramide. Inclusion of glucosylceramide in the culture medium prevented the stimulatory effect of PDMP on InsP3 formation. It is concluded that membrane glucosphingolipids may regulate phospholipase C activity.     

Calcium metabolism in intact isolated thymocytes.

Isolated rat thymocytes incubated under proper metabolic conditions extrude Ca2+ previously taken up under metabolically unfavourable conditions. The extrusion can be supported by both respiratory and glycolytic energy but glycolysis seems to be more efficient for this purpose. La3+ (50--200 micron) and the ionophore A 23187 inhibit cell Ca2+ extrusion. Ruthenium Red (1--100 micron) does not influence cell Ca2+ extrusion while it inhibits the in situ mitochondrial cation uptake. All the results are consistent with a cell regulation model of Ca2+ content in which both plasma membrane and mitochondria co-operate, acting in opposite directions, in order to decrease cytosolic Ca2+ concentration. The possibility of Na+-Ca2+ hetero-exchange participation to cell Ca2+ homeostasis regulation is also discussed.     

Origin of new glial cells in intact and injured adult spinal cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.     

Calcium dependence of exocytosis in lacrimal gland acinar cells

Simultaneous measurements of membranecapacitance and intracellular calcium concentration were used toexamine the calcium dependence of exocytosis in single acinar cellsfrom mouse lacrimal gland and to establish the quantitative relationbetween calcium concentration and rate of exocytosis. Application ofadrenergic or muscarinic agonists elevated intracellular calcium andevoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation waseliminated by internal dialysis with the calcium buffer EGTA, whichdemonstrated that the increase in intracellular calcium was necessaryfor agonist-evoked exocytosis. When internal calcium was elevated byapplication of the calcium ionophore ionomycin, exocytosis was evokedin the absence of agonist stimulation. Thus an increase inintracellular calcium was necessary and sufficient for exocytosis insingle acinar cells. The rate of change of membrane capacitanceincreased as approximately the third power of the calciumconcentration, which is similar to the dependence of exocytosis rate oncalcium concentration in other secretory cells.

     

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.