Comparative Effects of Chronic Exposure to Ethanol and Calcium Channel Antagonists on Calcium Channel Antagonist Receptors in Cultured Neural (PC12) Cells

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.     

Ontogeny of Calcium Antagonist Binding Sites in Chick Brain and Heart

The ontogeny of chick brain and heart ventricle calcium antagonist binding sites was determined, using [3H]nitrendipine ([3H]NDP), as the ligand. The binding of [3H]NDP to adult heart and brain was kinetically very similar, with the former displaying a KD of 0.28 +/- 0.02 nM and a Bmax of 138 +/- 17 fmol/mg protein, and the latter a KD of 0.30 +/- 0.02 nM and a Bmax of 160 +/- 12 fmol/mg protein. The binding site in both brain and heart was highly specific for dihydropyridine calcium antagonists, such as nifedipine, nimodipine, and nisoldipine, since these drugs were several orders of magnitude more potent as inhibitors of [3H]NDP binding than verapamil, methoxyverapamil, or diltiazem. The developmental appearance of [3H]NDP binding sites in brain was rather gradual, with adult levels being attained just prior to birth. This was in contrast to the profile in heart ventricle which showed essentially adult levels at seven days gestation. The acquisition of [3H]NDP binding sites in chick brain roughly paralleled the onset of neuronal maturation and functional activity. In both chick brain and heart, verapamil and methoxyverapamil were weak inhibitors of [3H]NDP binding. However, the inhibition of binding in both tissues was markedly biphasic, with only 50% of the binding sites being susceptible to inhibition by each agent, suggesting that multiple calcium antagonist binding sites may exist in both tissues.     

Gangliosides Stimulate Calcium Flux in Neuro-2A Cells and Require Exogenous Calcium for Neuritogenesis

The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal and neuroblastoma systems, but the mechanism is not understood. Involvement of Ca2+ is suggested by this study which demonstrates that treatment of Neuro-2A cells with bovine brain gangliosides (BBG) in Ca2(+)-depleted medium failed to produce neurite outgrowth. This was in contrast to treatment with retinoic acid or dibutyryl cyclic AMP which induced differentiation under the same conditions. Addition of BBG to Neuro-2A cells caused small, but significant, increases in both influx and efflux of Ca2+. It thus appears that although neuritogenesis can proceed by more than one mechanism, that induced by BBG requires exogenous Ca2+ and involves stimulation of Ca2+ flux.     

Enhancement of 45Ca2+ Binding to Acidic Lipids by Barbiturates, Diphenylhydantoin, and Ethanol

Abstract: Effects of barbiturates, diphenylhydantoin, and ethanol on ⁴⁵Ca²⁺ binding to acidic lipids have been examined in an organic solvent-aqueous partition system. Hexobarbital, pentobarbital, and phenobarbital, at concentrations of 0.3 and/or 0.6 mM, enhanced the binding of ⁴⁵Ca²⁺ to phosphatidic acid, phosphatidylserine, and sulfatide but not to phosphatidylinositol or cardiolipin. Diphenylhydantoin, 0.3 mM, enhanced ⁴⁵Ca²⁺ binding to phosphatidic acid and phosphatidylserine but not to sulfatide. Ethanol at 80 mM did not enhance ⁴⁵Ca²⁺ binding to phosphatidic acid, but ethanol decreased the binding to cardiolipin and increased it to sulfatide.     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Leptinotoxin-h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release

Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Calcium channels and their role in regenerative medicine

Stem cells hold indefinite self-renewable capability that can be differentiated into all desired cell types.Based on their plasticity potential,they are divided into totipotent(morula stage cells),pluripotent(embryonic stem cells),multipotent(hematopoietic stem cells,multipotent adult progenitor stem cells,and mesenchymal stem cells[MSCs]),and unipotent(progenitor cells that differentiate into a single lineage)cells.Though bone marrow is the primary source of multipotent stem cells in adults,other tissues such as adipose tissues,placenta,amniotic fluid,umbilical cord blood,periodontal ligament,and dental pulp also harbor stem cells that can be used for regenerative therapy.In addition,induced pluripotent stem cells also exhibit fundamental properties of self-renewal and differentiation into specialized cells,and thus could be another source for regenerative medicine.Several diseases including neurodegenerative diseases,cardiovascular diseases,autoimmune diseases,virus infection(also coronavirus disease 2019)have limited success with conventional medicine,and stem cell transplantation is assumed to be the best therapy to treat these disorders.Importantly,MSCs,are by far the best for regenerative medicine due to their limited immune modulation and adequate tissue repair.Moreover,MSCs have the potential to migrate towards the damaged area,which is regulated by various factors and signaling processes.Recent studies have shown that extracellular calcium(Ca²⁺)promotes the proliferation of MSCs,and thus can assist in transplantation therapy.Ca²⁺signaling is a highly adaptable intracellular signal that contains several components such as cell-surface receptors,Ca²⁺channels/pumps/exchangers,Ca²⁺buffers,and Ca²⁺sensors,which together are essential for the appropriate functioning of stem cells and thus modulate their proliferative and regenerative capacity,which will be discussed in this review.     

Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells

Interaction of Li+ with the voltage-dependent Na+ channel has been analyzed in neuroblastoma X glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage-dependent Na+ channels. Veratridine and aconitine increased the uptake of Li+ to the same degree (EC50 30 microM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine-stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 micrograms/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage-dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.     

Leptinotoxin-h Action in Synaptosomes, Neurosecretory Cells, and Artificial Membranes: Stimulation of Ion Fluxes

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Concanavalin a binding and Ca fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca²⁺ content of the cells. ⁴⁵Ca²⁺ fluxes were measured under conditions where artifacts due to Ca²⁺ binding to concanavalin A could be excluded. Both ⁴⁵Ca²⁺ influx into and efflux from these cells are significantly activated by the lectin. If ⁴⁵Ca²⁺ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in ⁴⁵Ca²⁺ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 μg/ml) no significant increase in ⁴⁵Ca²⁺ influx occurs but an increase in ⁴⁵Ca²⁺ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca²⁺ influx into the cells and that activation of Ca²⁺ efflux occurs as a response to an increase in the cytosolic Ca²⁺ activity. Thus, Ca²⁺ may well play a role in triggering lymphocyte activation.     

Regulation of Calcium Concentrations in Synaptosomes: α2-Adrenoceptors Reduce Free Ca2+ by Closure of N-Type Ca2+ Channels

The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K(+)-depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT (10.7 mM) was approximately 10,000 times higher than [Ca2+]i (118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaT was freely exchangeable. This pool was much larger than the free Ca2+ pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+ concentration in the buffer, suggesting that [Ca2+]i is not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time- and dose-dependent manner. Maximum inhibition (40% at 100 microM) occurred at 2 min and the IC50 was 80 nM. The reduction caused by clonidine (1 microM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+ pool size. The effects of clonidine (1 microM) on [Ca2+]i (26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+ concentration (0.1-1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of Calcium Channel Blocker Benidipine and Calmodulin Antagonist W7 on GDP-Binding Capacity of Brown Adipose Tissue in Mice

It has been suggested that increased dietary calcium intake can attenuate obesity. Calcium antagonists, such as benidipine, also have been shown to have an anti-obesity effect. However, the mechanism for calcium-related anti-obesity effect has not yet been established. A defective brown adipose tissue thermogenesis has been shown in obese rodents. This study was designed to examine the direct effects of calcium channel blocker benidipine and calmodulin antagonist W7 administration on the adaptive thermogenesis in brown adipose tissue taken from the genetically obese mice and their lean controls. The GDP binding to brown-fat cell mitochondria was used as a brown adipose tissue thermogenic index. The results show that benidipine treatment had no marked effect on brown-fat cell GDP-binding capacities in both obese and lean mice. However, GDP-binding capacities were significantly reduced in both obese and lean mice after the W7 administration. The results of this study support the previous finding that benidipine did not have direct thermogenic effect on brown adipose tissue and suggest that the change in intracellular calmodulin availability might contribute to the adaptive thermogenesis in brown adipose tissue.     

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

植物细胞中钙信号的时空多样性与信号转导

近年来,对钙信号的研究,包括对钙信号的产生,传导及最终靶蛋白的研究,越来越受到人们的重视,植物生长发育过程的信息传递,包括对各种内外刺激的反应都涉及到钙信号,钙信号的产生及传导是通过胞质自由钙离子的浓度变化来实现的,本文综述了胞质自由钙离子的测定,钙信号的时空多样性及钙信号的靶蛋白如CaM,Ca^2 依赖的蛋白激酶,钙调磷酸酶,磷脂酰肌醇－磷脂酶C等方面的一些最新进展,展望了今后钙信号研究的方向所用到的一些技术方法等。     

Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis

The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.     

Binding of Lanthanum Ions and Ruthenium Red to Synaptosomes and Its Effects on Neurotransmitter Release

A technique for studying the binding of La3+ to synaptosomes in a double-beam spectrophotometer, using murexide as indicator, is described. The binding of La3+ was very rapid and Scatchard plots revealed two components, with KD values of 0.6 and 27 microM in a Na+-free medium (sucrose medium) and 2.3 and 63 microM in an ionic medium containing 135 mM Na+. The binding of the cationic dye ruthenium red (RuR) showed only one site, with a KD of 3.7 microM. La3+ binding was partially inhibited by RuR and vice versa, and La3+ was also capable of partially displacing RuR previously bound to the synaptosomes, particularly in the sucrose medium. The release of labeled gamma-aminobutyric acid (GABA) stimulated by K+ depolarization was inhibited by La3+ concentrations at or above 1 microM, in the ionic medium, whereas in the sucrose medium 2.5 microM or higher La3+ concentrations notably stimulated the spontaneous release of both GABA and glutamic acid. It is concluded that La3+ and RuR share at least one type of binding site, which is probably the high-affinity La3+ site. Since both La3+ and RuR at low concentrations have been shown to block the depolarization-induced Ca2+ entry in synaptosomes, this site might be related to the voltage-dependent Ca2+ entry involved in neurotransmitter release.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.