The role of calcium in agonist-stimulated hydrolysis of phosphatidylinositol in mouse pancreas

The effects of Ca²⁺ on agonist-stimulated hydrolysis of myo-[2-³H]inostol-labelled phosphatidylinositol in mouse pancreas in vitro, were studied. The increase in cytosol Ca²⁺ concentration produced by the ionophore A23187 did not stimulate the breakdown of phosphatidylinositol. Cholecystokinin-octapeptide (CCK-8) stimulated the hydrolysis of phosphatidylinositol under conditions in which intracellular calcium stores were depleted. The breakdown of phosphatidylinositol was stimulated by bethanechol and CCK-8 in Ca²⁺-free Krebs solution, and the addition of Ca²⁺ to the medium potentiated the effects of these agonists. Lanthanum significantly reduced bethanechol and CCK-8 stimulated hydrolysis of phosphatidylinositol in Krebs solution, but was without effect in Ca²⁺-free Krebs solution. The results of this study support the proposal that PI hydrolysis does not occur as a result of Ca²⁺ mobilization and may be involved in Ca²⁺ gating in the pancreas.     

AXOPLASMIC TRANSPORT OF PROTEINS IN VITRO IN PRIMARY AFFERENT NEURONS OF FROG SPINAL CORD: EFFECT OF Ca2+ -FREE INCUBATION CONDITIONS

—The effects of Ca²⁺-free incubation medium on in vitro axoplasmic transport of proteins were studied in the central and peripheral branches of primary afferent spinal neurons of frog. Following exposure of dorsal root ganglia to [³H]leucine, the amount of radioactive protein transported along the axons during a subsequent 19 h period was decreased by approximately 60 per cent in preparations incubated in Ca²⁺-free, 1 mm -EGTA medium compared to those in normal medium. In similar Ca²⁺-free conditions the endogenous calcium levels were decreased to one-fourth the levels found following incubation in normal medium. Neither raising EGTA concentrations to 10 mm nor incubation in Ca²⁺-free medium prior to the [³H]leucine pulse were found to decrease the amount of transported protein in Ca²⁺-free medium by more than 70 per cent. The decrement in the amount of transported proteins did not appear to be due to an effect of Ca²⁺-free medium upon either the uptake of [³H]leucine into ganglion cells or upon the incorporation of radioactive amino acid into protein. The data are interpreted to suggest (i) that‘loading' of proteins onto the transport system is inhibited during Ca²⁺-free incubation and (ii) that the apparent transport of radioactive proteins during Ca²⁺-free incubation conditions might reflect proximo-distal movement of either microtubular protein or some other protein components of the transport system. It is proposed that calcium ions might function as reversible bonds between the transport system and‘transported' proteins.     

Flocculation of cell walls of brewer's yeast and effects of metal ions,protein-denaturants and enzyme treatments

Effects of metal ions, protein-denaturants and enzyme treatments on flocculation of cell walls of Beer Yeast IFO 2018 were investigated. Cell walls from flocculent cells grown in a complete medium were able to form flocs as were whole cells, but cell walls from non-flocculent cells, such as “Mg²⁺-deficient” cells, “early-phase” cells and “low-pH” cells, were not. The cell walls dispersed in distilled water reflocculated in solutions containing Ca²⁺ or other metal ions. Of the alkali metal ions tested, only Na⁺ inhibited flocculation of flocculent cell walls at a concentration more than 0.1 M. Ca²⁺ or Sn⁴⁺ was absolutely required for flocculation of cell walls in the physiological saline (NaCl, 150 mM), but the effect of Sn⁴⁺ seems rather non-specific, because it promoted flocculation of non-flocculent cell walls as well. Sr²⁺ and Ba²⁺ were antagonistic to Ca²⁺ and inhibited flocculation. Flocculation of cell walls was also depressed by high concentrations of protein-denaturants, e.g. urea and guanidine·HCl. Treatment with proteolytic enzymes deprived cell walls of floc-forming ability. Effect of metal ions, protein-denaturants and treatment with enzymes on the flocculation of intact cells was investigated as control. Since flocculating properties of cell walls were very similar to those of intact cells, flocculation must be an inherent property of cell walls.     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

Schistosoma mansoni: Calcium efflux and effects of calcium-free media on responses of the adult male musculature to praziquantel and other agents inducing contraction

When male Schistosoma mansoni were incubated in a Ca²⁺-free medium their responsiveness to the contracture inducing agents, praziquantel (PZ), dinitrophenol (DNP), 60 mM K⁺ (high K⁺), ouabain, and low temperature, was rapidly attenuated. After 5 min in a zero Ca²⁺ medium the responsiveness to PZ was reduced by 60% but a residual response of 20% remained even after 40 min in a calcium-free medium. The contracture induced by ouabain or low temperature was totally lost within 1 min exposure to a zero Ca²⁺ medium. The efflux of ⁴⁵Ca²⁺ from parasites incubated in a medium containing no Ca²⁺ closely parallels the drop in responsiveness of their musculature to high K⁺, DNP, and PZ. The total amount of Ca²⁺ in the parasite was reduced by only 30% after 60 min in zero Ca²⁺ medium. A relatively rapid exponential decline in muscle tension was noted when parasites, previously treated with PZ, high K⁺, or DNP, were transferred to a medium containing these agents but with no Ca²⁺. Parasites that had been contracted with ouabain or low temperature showed no significant relaxation 16 min after transfer to a zero Ca²⁺ medium. The ⁴⁵Ca²⁺ efflux from worms bathed in zero Ca²⁺ medium was not significantly altered by the presence of ouabain. These results suggest the presence of active Ca²⁺ transport at the level of the parasites' muscle membranes.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Histamine causes Ca entry via both a storemoperated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca²⁺] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca²⁺ enters the cell through a (capacitative) Ca²⁺ entry pathway activated as a consequence of intracellular Ca²⁺ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 μM) caused a rapid but transient increase in cytosolic [Ca²⁺] followed by a lower plateau that was sustained as long as external Ca²⁺ was present. In the absence of external Ca²⁺ only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca²⁺-free medium to deplete the internal Ca²⁺ stores, histamine caused no increase in cytosolic [Ca²⁺] when external Ca²⁺ was absent. Re-introduction of external Ca²⁺ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca²⁺] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H₁-receptor antagonist, mepyramine (2 μM). A comparison was made between store-dependent Ca²⁺ entry consequent upon store mobilization with histamine in Ca²⁺-free medium and plateau phase Ca²⁺ entry resulting from stimulation with histamine in Ca²⁺-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P ? 0.0001) at the same concentration of histamine (10 μM). It is concluded that histamine causes Ca²⁺ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca²⁺ entry through other pathways.     

CHARACTERIZATION OF PUTATIVE AMINO ACID TRANSMITTER RELEASE FROM SLICES OF RAT DENTATE GYRUS

Abstract— Superfused slices of the rat dentate gyrus were employed to study the release of GABA, glutamate and aspartate, which are considered strong neurotransmitter candidates in this region. The introduction of Ca²⁺ to a Ca²⁺-free superfusion medium containing a depolarizing agent augmented the efflux of all three amino acids. The response to application of Ca²⁺ nearly always occurred within 30 s, the shortest interval tested in these studies. The efflux rate reached a peak within 90 s and then declined to a level slightly greater than the prestimulation baseline. The failure to maintain the maximal rate with continued exposure to Ca²⁺ and depolarizing influences appeared not to result from a reduction in Ca²⁺ permeability caused by continuous depolarization. Ca²⁺ also stimulated the efflux of exogenously loaded radiolabeled GABA, glutamate and aspartate, but not proline. Exogenously loaded GABA was more readily released than endogenous GABA. Otherwise the effects of various treatments on their efflux rates were qualitatively similar. Mg²⁺ inhibited Ca²⁺-dependent efflux. Ba²⁺, but not Mg²⁺, stimulated amino acid efflux in the absence of Ca²⁺. Extracellular Na⁺ was not required to support Ca²⁺-dependent efflux. Addition of Ca²⁺ to a Ca²⁺-free medium in the absence of a depolarizing agent released GABA from the slices, but not glutamate or aspartate. K⁺-enriched medium and the depolarizing alkaloid, veratridine, stimulated both Ca²⁺-dependent and Ca²⁺-independent release processes. Na⁺-free medium enhanced the Ca²⁺-independent releasing action of elevated K⁺. Ca²⁺-independent release was inhibited by raising the Mg²⁺ concentration by 15 or 30 mM and appeared to be inhibited by Ca²⁺ as well. Amino acid output in the absence of Ca²⁺ is probably not directly related to transmission and is considered to result partially from a general increase in membrane permeability induced by depolarization in a Ca²⁺-free medium and partially from stimulation of carrier-mediated amino acid efflux. These results support previously suggested transmitter roles for GABA, glutamate and aspartate in the rat dentate gyrus.     

Cross-Talk of the Receptors for Bradykinin, Serotonin, and ATP Shown by Single Cell Ca2+ Responses Indicating Different Modes of Ca2+ Activation in a Neuroblastoma × Glioma Hybrid Cell Line

Abstract: Modes of Ca²⁺ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma × rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca²⁺ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca²⁺ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca²⁺-free medium abolished the Ca²⁺ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, Induces the release of Ca²⁺ from intracellular stores. Serotonin, in contrast, activates Ca²⁺ entry from the extracellular space. To investigate whether ATP releases Ca²⁺ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca²⁺. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca²⁺ response in Ca²⁺-free medium, but not in Ca²⁺-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca²⁺ stores not accessible to ATP that are refilled by extracellular Ca²⁺. Cyclic AMP and cyclic GMP apparently do not affect the Ca²⁺ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.     

Potentiation by ouabain of bradykinin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells: Evidence for involvement of Na+ influx associated with the bradykinin B2-receptor

The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na⁺-K⁺ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na⁺, as well as Ca²⁺, is involved in BK-receptor mediated CA secretion. BK (10⁻⁸–10⁻⁵M)-induced CA secretion was markedly potentiated by addition of ouabain (10⁻⁵M), was blocked by a BK-B₂ receptor antagonist, and was decreased in Ca²⁺-free medium. BK-induced increase in ⁴⁵Ca²⁺ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca²⁺-free medium in the presence or absence of ouabain and then kstimulated for 15 min with Ca²⁺-medium without BK or ouabain. Prior stimulation of the cells, BK induced ²²Na⁺ influx and increased Ca²⁺-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na⁺-free sucrose medium, the Ca²⁺-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na⁺ pump both increase the intracellular Na⁺ level, resulting in increase in Ca²⁺ influx and CA secretion.     

Possible role of phospholipase C in the induction of Ca2+-paradox in rat heart

In order to investigate the involvement of phosphoinositide-specific phospholipase C (PLC), an enzyme associated with phosphoinositide signal transduction pathway, for the occurrence of Ca²⁺-paradox (loss of contractile activity associated with contracture), rat hearts perfused with Ca²⁺-free medium (1 to 5 min) were reperfused (5 to 10 min) with medium containing 1.25 mM Ca²⁺. Crude membranes isolated from hearts perfused with Ca²⁺-free medium exhibited a significantly increased activity of PLC, whereas normal activity was detected in hearts reperfused with Ca²⁺-containing medium. A significant rise in PLC activity was observed at 1 min of Ca²⁺-free perfusion; maximal increase was seen at 4 min of Ca²⁺-free perfusion. Minimal concentration of Ca²⁺ in the perfusion medium required for showing an increase in PLC activity was 10 M, whereas that required for the occurrence of Ca²⁺-paradoxic changes in heart function upon reperfusion was 50M. Perfusion of the hearts with Ca²⁺-free medium in the presence of low Na⁺ or at low temperature, which prevents the occurrence of Ca²⁺-paradox upon reperfusion, did not prevent the increase in PLC activity. An increase during Ca²⁺-free perfusion similar to that seen for PLC was also observed for two other enzymes, namely the phosphatidylinositol (PI) 4-kinase and the PI-4-monophosphate (PIP) 5-kinase, which synthesize the PLC substrate, phosphatidylinositol 4,5-bisphosphate (PIP₂). No alteration of the alpha-adrenoreceptors was observed after 5 min of Ca²⁺-free perfusion. On the other hand, the observed changes in PLC activity during Ca²⁺-free perfusion appear to be due to some redistribution of the enzyme in the myocardium. These results suggest a possible role of the phosphoinositide/PLC pathway in the induction of Ca²⁺-paradox via mechanisms which do not appear to be associated with changes in the characteristics of alpha-adrenergic receptors. (Mol Cell Biochem121: 181–190, 1993)     

Comparative analysis of Ca2+-signaling in brown preadipocytes of the ground squirrel (Spermophillus undulatus) and mouse

We compared the Ca²⁺ response to noradrenalin, tapsigargin, thimerosal, and ionomycin in brown preadipocytes of the ground squirrel (Spermophillus undulatus) and mouse. The ground-squirrel brown preadipocytes did not respond to noradrenalin in concentrations within the physiological range. Stimulation of the plasma membrane Ca²⁺-channels with thimerosal showed a considerable reduction of the calcium entry in cell precursors of the both species. Intracellular calcium stores liberated in the preadipocytes of the both species by tapsigargin and ionomycin in Ca²⁺-free medium were insignificant. Ca²⁺-entry in preadipocytes was not activated by the intracellular Ca²⁺-store depletion. The Ca²⁺ response of the ground squirrel brown preadipocytes was independent of an animal’s physiological state or annual seasons. Brown preadipocytes of ground squirrels (Spermophillus undulatus) may be considered to be high ionomycin-resistant cells with reduced Ca²⁺-signaling systems.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 μM) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) from 72 ± 5 to 3042 ± 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca²⁺]_i, the effect of LPC was examined in the Ca²⁺-free solution containing EGTA. Under the Ca²⁺ -free conditions, LPC did not increase [Ca²⁺]_i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca²⁺-present condition. Addition of ryanodine (10 μM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca²⁺-free and Ca²⁺ -present conditions. Preincubation of the myocytes with d-propranolol (50 μM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca²⁺ -free and Ca²⁺ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca²⁺]_i, and the Ca²⁺ -independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

Control of the onset of migration of neural crest cells in avian embryos

Summary To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of Ca^{+ +} and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3–8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca^{+ +}- and Mg^{+ +}-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca^{+ +}-free medium duplicated the effects of CMF, but neither Mg^{+ +}-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane. Ca^{+ +}-channel antagonists D600 and Nifedipine, without any exernal Ca^{+ +}-depletion.The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca^{+ +}-depletion inactivates Ca^{+ +}-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca^{+ +}-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca^{+ +}-channels suggest that Ca^{+ +} levels may be involved in regulating this system.     

Participation of the microtubular-microfilamentous system on intracellular Ca transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[¹⁴C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca²⁺. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca²⁺, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca²⁺ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca²⁺. In addition, an increase in cytosolic Ca²⁺ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca²⁺ increase induced by A23187 in a Ca²⁺-free medium. On the other hand, in a Ca²⁺-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca²⁺ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca²⁺ uptake. These observations indicate that intracellular Ca²⁺ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca²⁺ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca2+ influx

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca²⁺) concentration ([Ca²⁺]_i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca²⁺]_i via blocking the influx of extracellular Ca²⁺ or chelating ooplasmic free Ca²⁺. After IVM, cumulus–oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca²⁺]_i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus–oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca²⁺]_i was significantly decreased. When oocytes were cultured in Ca²⁺-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca²⁺]_i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca²⁺]_i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca²⁺ or reducing ooplasmic free Ca²⁺. 1-Octanol, BAPTA-AM, or Ca²⁺-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.     

Angiotensin II causes calcium entry into bovine adrenal chromaffin cells via pathway(s) activated by depletion of intracellular calcium stores

The characteristics and properties of the increase in cytosolic [Ca²⁺] that occurs in bovine adrenal medullary chromaffin cells on exposure to angiotensin II have been investigated. In fura-2 loaded cells exposure to a maximally effective concentration of angiotensin II (100 nM) caused a rapid, but transient increase in cytosolic [Ca²⁺] followed by a lower plateau that was sustained as long as external Ca²⁺ was present. In the absence of external Ca²⁺ only the initial brief transient was observed. In cells previously treated with thapsigargin in Ca²⁺-free medium to deplete the internal Ca²⁺ stores, angiotensin II caused no increase in cytosolic [Ca²⁺] when external Ca²⁺ was absent. Reintroduction of external Ca²⁺ to thapsigargin-treated, store-depleted cells caused a sustained increase in cytosolic [Ca²⁺] that was not further increased upon exposure to angiotensin II. Analysis of the data suggests that in bovine chromaffin cells angiotensin II causes Ca²⁺ entry via a pathway(s) activated as a consequence of internal store mobilization, and entry through this pathway(s) forms the majority of the sustained Ca²⁺ influx evoked by angiotensin II.