ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.     

Hydrogen sulfide evokes neurite outgrowth and expression of high-voltage-activated Ca2+ currents in NG108-15 cells: involvement of T-type Ca2+ channels

We investigated if stimulation of T-type Ca²⁺ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5–13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca²⁺, and by zinc chloride at a concentration known to selectively inhibit Ca_v3.2 isoform of T-type Ca²⁺ channels, but not Ca_v3.1 or Ca_v3.3. Further, l -ascorbate, recently proven to selectively inhibit Ca_v3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H₂S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca²⁺ channels, especially Ca_v3.2.     

Possible Regulation of Caffeine-Induced Intracellular Ca2+ Mobilization by Intracellular Free Na+

To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in.     

Ca2+ - and Nitric Oxide-Dependent Stimulation of Cyclic GMP Synthesis in Neuronal Cell Line Induced by P2-Purinergic/Pyrimidinergic Receptor

Abstract: The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ～1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′-(γ-thio)triphosphate > UTP = 2-methylthio-ATP > ADP ? CTP, AMP, α,β-methylene-ATP, 2′- and 3′-O-(4-benzoylbenzoyl)ATP. The EC₅₀ of 1 ± 0.2 µM for UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P_2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC₅₀ = 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (IC₅₀ = 20–30 µM). Pretreatment of cells with the Ca²⁺ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca²⁺ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC₅₀ for nitroarginine = 1 µM) or by hemoglobin (IC₅₀ = 2 µM). This indicates that ATP/UTP via a P₂-receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca²⁺ level, mostly due to release of Ca²⁺ from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross-desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ～40% only. This indicates a heterologous cross-desensitization predominantly in one direction (bradykinin ? ATP).     

δ-Opioids Stimulate Inositol 1,4,5-Trisphosphate Formation, and so Mobilize Ca2+ from Intracellular Stores, in Undifferentiated NG108-15 Cells

Abstract: δ-Opioids mobilize Ca²⁺ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [d -Pen^2,5]enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [d -Pen^2,5]enkephalin caused a dose-dependent (EC₅₀ = 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7 ± 1.2 pmol/mg of protein with 1 µM, n = 9) at 30 s and returned to basal levels (10.6 ± 0.9 pmol/mg of protein, n = 9) within 4–5 min. This response was fully naloxone (1 µM) reversible and pertussis toxin (100 ng/ml for 24 h) sensitive. Preincubation with Ni²⁺ (2.5 mM) or nifedipine (1 µM) had no effect on the [d -Pen^2,5]enkephalin (1 µM)-induced inositol 1,4,5-trisphosphate response, and K⁺ (80 mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca²⁺ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 µM) enhanced, whereas acute exposure to phorbol 12,13-dibutyrate (1 µM) abolished, the [d -Pen^2,5]enkephalin (0.1 µM)-induced inositol 1,4,5-trisphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [d -Pen^2,5]Enkephalin also dose-dependently (EC₅₀ = 2.8 nM) increased the intracellular [Ca²⁺], which was maximal (24 nM increase with 1 µM, n = 5) at 30 s. This close temporal and dose-response relationship strongly suggests that δ-opioid receptor-mediated increases in intracellular [Ca²⁺] results from inositol 1,4,5-trisphosphate-induced Ca²⁺ release from intracellular stores, in undifferentiated NG108-15 cells.     

Chronic Exposure of NG 108-15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide-Binding Proteins Gi and GO

We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go.     

Prostaglandin Induces Ca2+ Influx and Cyclic GMP Formation in Mouse Neuroblastoma × Rat Glioma Hybrid NG108–15 Cells in Culture

Various prostaglandins (PGs) (10 nM-30 microM) were added to NG108-15 cells in culture, and changes in the levels of intracellular cyclic GMP and Ca2+ were investigated. Exposure of the cells to PGF2 alpha, PGD2, and PGE2 (10 microM) transiently increased the cyclic GMP content 7.5-, 3.9-, and 3.1-fold, respectively. Furthermore, the increased levels of cyclic GMP correlated well with the rise in cytosolic free Ca2+ concentrations induced by the PGs. Other PGs (10 microM), including metabolites and synthetic analogs, which had no effect on intracellular Ca2+, failed to increase the cyclic GMP content in the cells. When extracellular Ca2+ was depleted from the culture medium, the PG-induced increase in cyclic GMP level was almost completely abolished. In addition, treatment of the cells with quin 2 tetraacetoxymethyl ester dose-dependently inhibited the PG-induced cyclic GMP formation. The increase in cyclic GMP content caused by treatment of the cells with a high K+ level (50 mM) was completely blocked by voltage-dependent Ca2+ entry blockers, such as verapamil (10 microM), nifedipine (1 microM), and diltiazem (100 microM); however, the PG (10 microM)-induced increase in cyclic GMP content was not affected by such Ca2+ entry blockers. These findings indicate that PG-induced cyclic GMP formation may require the rise in intracellular Ca2+ level and that the voltage-dependent Ca2+ channels may not be involved in the PG-induced rise in Ca2+ content.     

Differential subcellular localization of two dopamine D2 receptor isoforms in transfected NG108-15 cells

The dopamine D2 receptor (D2R) is target for antipsychotic drugs and associated with several neuropsychiatric disorders. D2R has a long third cytoplasmic loop and a short carboxyl-terminal cytoplasmic tail. It exists as two alternatively spliced isoforms, termed D2LR and D2SR, which differ in the presence and absence, respectively, of a 29 amino acid insert in the third cytoplasmic loop. To evaluate the differential roles of the two D2R isoforms, we transfected both isoforms into NG108-15 cells and observed their subcellular localization by a confocal laser scanning light microscope. D2SR was predominantly localized at the plasma membrane, whereas D2LR was mostly retained in the perinuclear region around the Golgi apparatus. Using a yeast two hybrid system with a mouse brain cDNA library and coimmunoprecipitation assay, we found that heart-type fatty acid binding protein (H-FABP) interacts with D2LR but not with D2SR. H-FABP is a cytosolic protein involved in binding and transport of fatty acids. Overexpressed H-FABP and endogenous H-FABP were colocalized with the intracellular D2LR in NG108-15 cells. Furthermore, in the rat striatum, H-FABP was detected in the D2R-expressing neurons. From these results, H-FABP is associated with D2LR, and may thereby modulate the subcellular localization and function of D2LR.     

Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide- and ATP-Phosphate Turnover in 32P-Labeled Rabbit Iris Smooth Muscle

Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine-stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [gamma-32P]ATP. Incorporation of 32P1 into ATP and phosphoinositides is time-dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Calcium ion, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P-labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca2+, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P-labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. Furthermore, the neurotransmitter stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P-labeled muscle only in the presence of both Na+ and Ca2+. These data provide additional support for the concept that in the rabbit iris receptor-activated Ca2+ fluxes mediate or precede the effects of alpha-adrenergic and cholinergic muscarinic agents on phosphoinositide breakdown into 1,2-diacylglycerol and inositol phosphates and that restoration of the polar head groups to the 1,2-diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+ -pump mechanism.     

Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5-HT3 Receptor-Mediated Cation Permeability in NG 108-15 Cells

Abstract: In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5-hydroxy-tryptamine (5-HT)] and substance P induces the opening of a cation permeability monitored by the influx of [¹⁴C]-guanidinium. The serotoninergic component of this cation permeability Is linked to 5-HT₃ receptor activation, whereas the substance P component probably involves an “N-terminal-dependent substance P receptor.” In this study, [¹⁴C]guanidinium influx triggered by 1 μM 5-HT plus 10 μM substance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω-conotoxin, as expected from a process independent of voltage-sensitive sodium and calcium channels. In contrast, [¹⁴C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis-O-aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [¹⁴C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [¹⁴C]guanidinium uptake was markedly increased even in the absence of 5-HT and/or substance P. Conversely, [¹⁴C]guanidinium influx due to the latter substances could be reversibly and dose-dependently blocked by various drugs that possess calmodulin-antagonizing properties. These results strongly suggest that the cation permeability opened by 5-HT and substance P in NG 108–15 cells involves a calcium/calmodulin-dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12-O-tetradeca-noylphorbol 13-acetate did not alter [¹⁴C]guanidinium influx in NG 108–15 cells exposed to 5-HT and substance P., this process probably does not involve the calcium-dependent nitric oxide pathway and protein kinase C activation.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Coupling of the Cloned μ-Opioid Receptor with the ω-Conotoxin-Sensitive Ca2+ Current in NG108-15 Cells

Abstract: Voltage-dependent Ca²⁺ currents were measured in NG108-15 neuroblastoma × glioma hybrid cells transformed to express the rat μ-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba²⁺ as charge carrier. A μ-opioid receptor-selective agonist, [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin caused significant inhibition of voltage-dependent Ca²⁺ currents in μ-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a δ-opioid receptor-selective agonist, [ d -penicillamine², d -penicillamine⁵]enkephalin, induced inhibition of voltage-dependent Ca²⁺ currents in both control and μ-receptor-transformed cells, which is mediated by the δ-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca²⁺ currents induced by [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin and [ d -penicillamine², d -penicillamine⁵]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω-conotoxin GVIA. These results indicate that the μ-opioid receptor expressed from cDNA functionally couples with ω-conotoxin-sensitive N-type Ca²⁺ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Amplification of depolarization-induced and ryanodine-sensitive cytosolic Ca2+ elevation by synthetic carbocyclic analogs of cyclic ADP-ribose and their antagonistic effects in NG108-15 neuronal cells

We synthesized analogs modified in the ribose unit (ribose linked to N1 of adenine) of cyclic ADP-ribose (cADPR), a Ca2+-mobilizing second messenger. The biological activities of these analogs were determined in NG108-15 neuroblastoma x glioma hybrid cells that were pre-loaded with fura-2 acetoxymethylester and subjected to whole-cell patch-clamp. Application of the hydrolysis-resistant cyclic ADP-carbocyclic-ribose (cADPcR) through patch pipettes potentiated elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) at the depolarized membrane potential. The increase in [Ca2+]i evoked upon sustained membrane depolarization was significantly larger in cADPcR-infused cells than in non-infused cells and its degree was equivalent to or significantly greater than that induced by cADPR or beta-NAD+. 8-Chloro-cADPcR and two inosine congeners (cyclic IDP-carbocyclic-ribose and 8-bromo-cyclic IDP-carbocyclic-ribose) did not induce effects similar to those of cADPcR or cADPR. Instead, 8-chloro-cADPcR together with cADPR or cADPcR caused inhibition of the depolarization-induced [Ca2+]i increase as compared with either cADPR or cADPcR alone. These results demonstrated that our cADPR analogs have agonistic or antagonistic effects on the depolarization-induced [Ca2+]i increase and suggested the presence of functional reciprocal coupling between ryanodine receptors and voltage-activated Ca2+ channels via cADPR in mammalian neuronal cells.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.