Relationship Between the Actions of Calcium Ions, Opioids and Prostaglandin E1 on the Level of Cyclic AMP in Neuroblastoma × Glioma Hybrid Cells

Abstract: Neuroblastoma × glioma hybrid cells increase their intracellular concentration of cyclic AMP in response to prostaglandin E₁ (PGE₁). This effect is inhibited by opioids. The response to PGE₁ is positively correlated with the concentration of Ca²⁺ in the incubation medium. The Ca²⁺ antagonists Co²⁺ and La³⁺, the Ca²⁺ chelator EGTA and a blocker of Ca²⁺ influx into cells, Segontin, inhibit the response to PGE₁. At low external concentrations of Ca²⁺ the response to PGE₁ is enhanced by the Ca²⁺ ionophore A23187. The effects of A23187 and Segontin point to a cytosolic site of Ca²⁺ action. Lack of Ca²⁺ reduces the level of cyclic AMP even in the absence of PGE₁ and the presence of an inhibitor of cyclic AMP phosphodiesterase. Ca²⁺ is required even for an increase in the level of cyclic AMP in cells pretreated with cholera toxin. The increases in level of cyclic AMP evoked by PGE, in a neuroblastoma and by PGE₁ or noradrenaline in a glioma cell line do not depend on Ca²⁺. The response of the hybrid cells to the opioid leucine-enkephalin appears not to rely on the presence of Ca²⁺. Even changing the intracellular concentration of Ca²⁺ by the ionophore A23187 does not alter the effect of the opioid. The analogy between opioids and lack of Ca²⁺ in the short-term (minutes) experiments mentioned holds also for long-term (hours) experiments. Cells chronically exposed to opioids or to low concentrations of Ca²⁺ display an enhanced maximal response to PGE₁.     

The δ-Opioid Receptor Regulates Activity of Ryanodine Receptors in the Human Neuroblastoma Cell Line SK-N-BE

Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) either by regulating plasma membrane Ca²⁺-channel activity or by mobilizing intracellular Ca²⁺ stores. The present report documents the [Ca²⁺]_i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing δ-opioid receptors. In the presence, as well as in the absence, of extracellular Ca²⁺, opioid agonists enhanced significantly [Ca²⁺]_i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ stores, acted only in the presence of extracellular Ca²⁺. The opioid-induced increase in [Ca²⁺]_i was not affected by treatments modifying the trimeric G_i, G_o, and G_s protein transduction mechanisms or the activity of adenylyl cyclase. The Ca²⁺-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca²⁺]_i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, δ-opioid receptors mobilize Ca²⁺ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of G_i/G_o and G_s proteins and protein kinase A activation.     

Methacholine-induced cGMP production is regulated by nitric oxide generation in rabbit submandibular gland cells

Guanosine 3′,5′-monophosphate (cGMP) is an intracellular messenger in various kinds of cell. We investigated the regulation of cGMP production by nitric oxide (NO) in rabbit submandibular gland cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose- and time-dependent manner, but the α-agonist phenylephrine, substance P and the β-agonist isoproterenol failed to evoke cGMP production. In fura-2-loaded cells, methacholine induced an increase in intracellular Ca²⁺ ([Ca²⁺]_i) in a concentration-dependent manner, which was similar to that for cGMP production. When the external Ca²⁺ was chelated with EGTA, methacholine failed to induce cGMP production. Ca²⁺ ionophore A23187 and thapsigargin, which induce the increase in [Ca²⁺]_i without activation of Ca²⁺-mobilizing receptors, mimicked the effect of methacholine. cGMP production induced by methacholine, A23187 and thapsigargin was clearly inhibited by N^G-nitro- -arginine methylester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS). S-Nitroso-N-acetyl- -penicillamine (SNAP), a NO donor, induced cGMP formation. In the lysate of rabbit submandibular gland cells, Ca²⁺-regulated nitric oxide synthase activity was detected. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is regulated by NO generation via the increase in [Ca²⁺]_i.     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line

The stimulation of IP₃ production by muscarinic agonists causes both intracellular Ca²⁺ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na⁺ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na⁺ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na⁺ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent K _D of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca²⁺ influx and electrical excitability. In N1E-115 cells, Ca²⁺ entry by this pathway is necessary to refill the IP₃-sensitive intracellular Ca²⁺ pool during repeated stimulation and CNG channels may play a similar role in other neurons.     

Hyperpolarization- and Depolarization-activated Ca2+ Currents in Paramecium Trigger Behavioral Changes and cGMP Formation Independently

Using 5% ethanol as a deciliating agent, 20 mm colchicine to prevent reciliation and 1 mm amiloride to affect ion fluxes in Paramecium we examined the compartmentation and function of Ca²⁺ fluxes employing the biosynthesis of cGMP and the stereotypic swimming behavior as indicators for Ca²⁺ entry. As a function of extracellular Ca²⁺ Paramecia responded to colchicine and amiloride with a short-lived ciliary augmentation (fast swimming) which indicated hyperpolarization, and formation of cGMP, i.e., the reported hyperpolarization-activated Ca²⁺ inward current in the somatic membrane is coupled to intracellular generation of cGMP. This is comparable to the coupling of the depolarization-activated, ciliary Ca²⁺ inward current and ciliary cGMP formation. Ethanol-deciliated cells and ethanol-treated, yet ciliated control cells did not respond to a depolarization with backward swimming or formation of cGMP. Both responses recovered with similar kinetics. A persistent effect of an ethanol exposure on the axonemal apparatus or on guanylyl cyclase activity of ciliated control cells was excluded using permeabilized cells and cell-free enzyme, respectively. Further, in the presence of 20 mm colchicine ethanol-treated cells only recovered the depolarization-dependent avoiding reaction whereas the formation of cGMP remained depressed, i.e., the drug dissected both responses. Similarly, ethanol exposure of Paramecia did not affect the fast swimming response towards the hyperpolarizing agent amiloride whereas the cGMP formation was abrogated and recovered over a period of 7 hr, i.e., amiloride dissected the hyperpolarization-elicited behavioral response from the intracellular cGMP formation. The data demonstrate that in Paramecium depolarization- and hyperpolarization-stimulated behavioral responses and cGMP formation are not coupled. The behavioral changes are triggered by smaller Ca²⁺ inward currents than the formation of intracellular cGMP. Received: 8 August 1996/Revised: 15 November 1996     

Muscarinic Acetylcholine Receptors Potentiate 5′-Adenosine Monophosphate-Activated Protein Kinase Stimulation and Glucose Uptake Triggered by Thapsigargin-Induced Store-Operated Ca<Superscript>2+</Superscript> Entry in Human Neuroblastoma Cells

The 5′-adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of the cellular energy metabolism and may induce either cell survival or death. We previously reported that in SH-SY5Y human neuroblastoma cells stimulation of muscarinic acetylcholine receptors (mAChRs) activate AMPK by triggering store-operated Ca²⁺ entry (SOCE). However, whether mAChRs may control AMPK activity by regulating additional mechanisms beyond SOCE remains to be investigated. In the present study we examined the effects of mAChRs on AMPK when SOCE was induced by the sarco–endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin. We found that in SH-SY5Y cells depleted of Ca²⁺ by thapsigargin, the re-addition Ca²⁺ to the medium stimulated AMPK phosphorylation at Thr172, which is required for full kinase activity. This response occurred through SOCE, as it was blocked by either the SOCE modulator 2-aminoethoxydiphephenyl borate, knockdown of the SOCE molecular component STIM1, or inhibition of Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase β (CaMKKβ). In thapsigargin-pretreated cells, stimulation of pharmacologically defined M₃ mAChRs potentiated SOCE-induced AMPK activation. This potentiation did not involve an increased Ca²⁺ influx, but was associated with CaM mobilization from membrane to cytosol, increased CaM/CaMKKβ interaction, and enhanced CaMKK stimulation by thapsigargin-induced SOCE. In thapsigargin-pretreated cells Ca²⁺ re-addition stimulated glucose uptake and increased the membrane expression of the glucose transporter GLUT1. Both responses were significantly potentiated by mAChRs. These data indicate that in human neuroblastoma cells mAChRs up-regulate AMPK and the downstream glucose uptake by triggering not only SOCE but also CaM translocation and enhanced formation of active CaM/CaMKKβ complexes.     

Stimulatory effects of opioids on transmitter release and possible cellular mechanisms: Overview and original results

Opiates and opioid peptides carry out their regulatory effects mainly by inhibiting neuronal activity. At the cellular level, opioids block voltage-dependent calcium channels, activate potassium channels and inhibit adenylate cyclase, thus reducing neurotransmitter release. An increasing body of evidence indicates an additional opposite, stimulatory activity of opioids. The present review summarizes the potentiating effects of opioids on transmitter release and the possible cellular events underlying this potentiation: elevation of cytosolic calcium level (by either activating Ca²⁺ influx or mobilizing intracellular stores), blockage of K⁺ channels and stimulation of adenylate cyclase. Biochemical, pharmacological and molecular biology studies suggest several molecular mechanisms of the bimodal activity of opioids, including the coupling of opioid receptors to various GTP-binding proteins, the involvement of different subunits of these proteins, and the activation of several intracellular signal transduction pathways. Among the many experimental preparations used to study the bimodal opioid activity, the SK-N-SH neuroblastoma cell line is presented here as a suitable model for studying the complete chain of events leading from binding to receptors down to regulation of transmitter release, and for elucidating the molecular mechanism involved in the stimulatory effects of opioid agonists. Special issue dedicated to Dr. Eric J. Simon.     

Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G _j) and cytoplasmic concentration of free Ca²⁺ ([Ca²⁺]_i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G _j varied for different cell pairs. One population exhibited a bell-shape dependence of G _j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca²⁺]_i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca²⁺, blockage of K⁺ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca²⁺ or blockage of K⁺ efflux, formation of whole-cell configuration generated a Ca²⁺ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca²⁺ release from intracellular Ca²⁺ stores, which activates sustained Ca²⁺ influx, K⁺ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Action of Iron Nitrosyl Complexes,NO Donors,on the Activity of Sarcoplasmic Reticulum Ca<Superscript>2+</Superscript>-ATPase and Cyclic Guanosine Monophosphate Phosphodiesterase

The effect of iron nitrosyl complexes, NO donors, of a general formula [Fe₂(L)₂(NO)₄] with functional sulfur-containing ligands (L-3-nitro-phenol-2-yl, 4-nitro-phenol-2-yl, or 1-methyl-tetrazol-5-yl) on the activity of sarcoplasmic reticulum Ca²⁺-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) was studied. The test complexes uncoupled the hydrolytic and transport functions of Ca²⁺- ATPase, thus disturbing the balance of Ca²⁺ ions in cells, which may affect the formation of thrombi and adhesion of metastatic cells to the endothelium of capillaries. They also inhibited the activity of cGMP PDE, thereby contributing to the accumulation of the second messenger cGMP. The studied iron nitrosyl complexes can be considered as potential drugs.     

Functional effects of expression of hslo Ca activated K channels in cultured macrovascular endothelial cells

The aim of the present study is to elucidate the effects of the expression of large conductance Ca²⁺ activated K⁺ channels (BK_Ca) in an endothelial cell type normally lacking this channel. The human homologue hslo of BK_Ca was expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which have no endogenous BK_Ca. Membrane potential, ionic currents and Ca²⁺ signals were investigated in non-transfected and transfected cells using a combined patch clamp and Fura-2 fluorescence technique. In non-transfected control CPAE cells, ATP evoked a Ca²⁺ activated CI⁻ current (I_cl,ca). The most prominent current component during ATP stimulation in hslo expressing cells was conducted 13K Ca which resulted in a pronounced transient hyperpolarization. This hyperpolarization, which was absent in non-transfected cells, was enhanced if I_Cl,Ca was blocked with niflumic acid. The sustained component of the Ca²⁺ response during ATP stimulation was significantly larger in hslo transfected cells than in non-transfected cells. This plateau level correlated well with the corresponding effects of ATP on the membrane potential, indicating that the expression of cloned BK_Ca exerts a positive feedback on Ca²⁺ signals in endothelial cells by counteracting the negative (depolarizing)effect of stimulation of Ca²⁺-activated CI⁻ channels.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Cyclosporin a Potentiates Receptor-Activated [Ca2+]c Increase

Abstract

The use of the immunosuppressant cyclosporin A (CsA) is frequently associated with hypertension. Drug-induced local vasoconstriction appears to be responsible for this effect. Using fura-2 and ⁴⁵Ca²⁺ efflux techniques, we have examined variations in the cytosolic calcium concentration ([Ca²⁺]_c) in rat aortic smooth muscle cells and have shown that increases in [Ca²⁺]_c after [Arg⁸]vasopressin, serotonin, endothelin-1 or angiotensin II stimulation were potentiated after preincubation of cells with CsA. This effect was independent of cyclophilin or calcineurin inhibition by CsA. Measurements of inositol phosphates (InsP_n) after agonist stimulation showed that CsA also potentiated their formation. As for ⁴⁵Ca²⁺ efflux this effect was not related to cyclophilin or calcineurin inhibition. Direct stimulation of G proteins with aluminium tetrafluoride induced an increase in InsP_n formation and ⁴⁵Ca²⁺ efflux. Neither of these responses was potentiated by CsA. These results indicate that CsA acts on a target upstream of G protein activation, possibly at the receptor level, resulting in a potentiation of InsP_n formation and subsequent calcium increase.     

The effects of CA2+ during the elicitation of shikonin derivatives inOnosma paniculatum cells

Summary A fungal elicitor extracted fromAspergillus oryzae (Ahlb.) Cobn mycelia promoted the production of shikonin derivatives inOnosma paniculatum Bur et Franch cell suspension cultures. Elicitor treatment also increased Ca²⁺ concentration in RM₉ medium, which could be measured earlier than the elicited increase of shikonin formation. Several reagents known to induce Ca²⁺-influx and increase the intracellular-free Ca²⁺ level, such as the addition of Ca (NO₃)₂·4H₂O, the Ca²⁺ ionophore A₂₃₁₈₇, and abscisic acid (ABA), appreciably suppressed the elicitor-promoted shikonin formation inOnosma cells. In contrast, the decrease of intracellular-free Ca²⁺ level by the specific Ca²⁺-chelator ethylene glycol bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or the Ca²⁺—channel blocker, verapamil, enhanced the biosynthesis of shikonin even in the absence of elicitor. Treatment of cells with trifluoperazine (TFP) also stimulated shikonin formation inOnosma cell cultures. A rapid and transient drop of free Ca²⁺ level in one protoplast was directly determined after the addition of elicitor toOnosma cell cultures. The inhibitory effect on shikonin formation by ABA was largely on account of its ability to restore the intracellular Ca²⁺ level lowered by the elicitor. These results suggest that Ca²⁺ play a significant role in an early stage of the elicitation process ofOnosma cells. The rapid drop of cytoplasmic Ca²⁺ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives.     

Neuroblasts-glia interaction. The effect of co-cultivation upon ecto-ATPase activity of neuroblastoma and glioma cells.

The effect of cell contact and cell medium upon the ecto-enzymes, Mg²⁺- and Ca²⁺-dependent ATPase and 5′-nucleotidase were studied in nervous system cells in tissue culture. Conditions were worked out for co-culture and rseparation of glioblastoma and neuroblastoma cells so that the effects upon each of the co-cultured cell lines after interaction of these cells could be reliably determined. Co-cultivation of mouse neuroblastoma and glioma cell lines markedly enhanced Mg²⁺- and Ca²⁺-dependent ecto-ATPase activity. Evidence was obtained which indicates that increase in ecto-ATPase of co-cultured neuro- and glioblastoma cells occurs in both cell types. Ecto-ATPase was 500% of the original level in clonal line NN astroblasts after co-culture with M1 neuroblasts. This activity decreased over 50 transfers during the period of about a year. Increase in ecto-ATPase and morphological differentiation of M1 neuroblastoma cells after co-culture with NN astroblasts could also be brought about simply by treatment with the medium from NN cell cultures. Co-cultivation of neuroblastoma and glioma cells does not change significantly the specific activity of ecto-5′-nucleotidase.     

Effect of Retinoic Acid on the Ca2+-Independent Phospholipase A2 in Nuclei of LA-N-1 Neuroblastoma Cells

LA-N-1 neuroblastoma cell cultures contain Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.     

Ion Channel Phenotype of Melanoma Cell Lines

Melanoma cells are transformed melanocytes of neural crest origin. K⁺ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (K_V), Ca²⁺-activated (K_Ca), and inwardly rectifying (K_IR) K⁺ channels; swelling-sensitive Cl⁻ channels (Cl_swell); voltage-gated Ca²⁺ channels (Ca_V) and Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores (CRAC); and voltage-gated Na⁺ channels (Na_V). The presence of Ca²⁺ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca²⁺] _i . The expression of K⁺ channels varied widely between different cell lines and was also influenced by culture conditions. K_IR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for K_IR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). K_Ca channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). K_Ca channels in C8146 and SK28 cells were sensitive to CTX (K _d = 4 nm), but were unaffected by apamin. K_V channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Cl_swell channel. Cl_swell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl⁻). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996     

Nitric oxide augments single Ca2+ channel currents via cGMP-dependent protein kinase in Kenyon cells isolated from the mushroom body of the cricket brain

Behavioral and pharmacological studies in insects have suggested that the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is involved in the formation of long-term memory (LTM) associated with olfactory learning. However, the target molecules of NO and the downstream signaling pathway are still not known. In this study, we investigated the action of NO on single voltage-dependent Ca²⁺ channels in the intrinsic neurons known as Kenyon cells within the mushroom body of the cricket brain, using the cell-attached configuration of the patch-clamp technique. Application of the NO donor S-nitrosoglutathione (GSNO) increased the open probability (NP_O) of single Ca²⁺ channel currents. This GSNO-induced increase was blocked by ODQ, a soluble guanylate cyclase (sGC) inhibitor, suggesting that the NO generated by GSNO acts via sGC to raise cGMP levels. The membrane-permeable cGMP analog 8-Bro-cGMP also increased the NP_O of single Ca²⁺ channel currents. Pretreatment of cells with KT5823, a protein kinase G blocker, abolished the excitatory effect of GSNO. These results suggest that NO augments the activity of single Ca²⁺ channels via the cGMP/PKG signaling pathway. To gain insight into the physiological role of NO, we examined the effect of GSNO on action potentials of Kenyon cells under current-clamp conditions. Application of GSNO increased the frequency of action potentials elicited by depolarizing current injections, indicating that NO acts as a modulator resulting in a stimulatory signal in Kenyon cells. We discuss the increased Ca²⁺ influx through these Ca²⁺ channels via the NO/cGMP signaling cascade in relation to the formation of olfactory LTM.     

Muscarinic Receptor-Mediated Inositol 1,4,5-Trisphosphate Formation in SH-SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP₃) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ～60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca²⁺ with EGTA or inhibition of Ca²⁺ channels with Ni²⁺ showed that the plateau phase was dependent upon Ca²⁺ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca²⁺ from intracellular stores revealed that Ca²⁺ from this source was capable of supporting the peak phase of the InsP₃ response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca²⁺ concentration (20–100 nM) and suggests that under normal conditions, InsP₃ formation is enhanced by increases in cytosolic free Ca²⁺ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li⁺ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP₃ accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca²⁺ concentration amplify InsP₃ formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP₃ accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca²⁺ or protein kinase C activity.