Georg Reiser

Bradykinin causes a transient rise of intracellular Ca2+-activity in cultured neural cells

Abstract1.The concentration of intracellular free calcium ions was measured by spectrofluorometry in suspensions of quin2 loaded neural cell lines: neuroblastoma x glioma hybrid cells (clones 108CC15 and 108CC25) and polyploid rat glioma cells (clone C6-4-2).2.In these cells, bradykinin elicits a transient increase of the cytosolic Ca2+-activity in a dose-dependent manner (half-maximal effect at about 10 nM). The effect requires the presence of extracellular Ca2+. The time to peak is at most 10 s, the decay to the original level lasts 1 min and is followed by a period of 1–4 min during which Ca2+ activity is slightly below control value. Lys-bradykinin and Met-Lys-bradykinin evoke similar effects as bradykinin, but at concentrations 10 times lower.3.The cells desensitize upon repeated addition of bradykinin.4.Under the same conditions des-Arg1-bradykinin, des-Arg9-bradykinin, angiotensin II, substance P, apamin and histamine exerted no influence on the concentrations of free Ca2+.5.Similar to their effect in neural cell lines, bradykinin and Lys-bradykinin induce in primary astroglia-rich cultures from rat brain an increase in the concentration of cytosolic Ca2+ with the peak reached within 30 s and the decay to the original level lasting approximately 4 min.6.The significance of this effect of bradykinin on the cytosolic Ca2+-activity is discussed in relation to previous findings that bradykinin in the same cell lines induces a hyperpolarization, a rise of the cyclic GMP level and a breakdown of phosphoinositides.

Endothelin and a Ca2+ ionophore raise cyclic GMP levels in a neuronal cell line via formation of nitric oxide.

1 The vasoconstrictor peptide endothelin‐1 caused a fast, transient rise in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels in a neuronal cell line (mouse neuroblastoma x rat glioma hybrid cells 108CC15). The mechanism of activation of guanylate cyclase by endothelin‐1 was investigated. The endothelin‐1‐induced rise depended on the release of internal Ca2+. 2 The stimulation of cyclic GMP synthesis induced by endothelin‐1 was suppressed after preincubating the cells in medium containing haemoglobin (IC50 3 μm). Similarly, pretreatment of the cells with the l‐arginine analogues, l‐canavanine (IC50 60 μm) or NG‐monomethyl‐l‐arginine (IC50 2.5 μm), inhibited the cyclic GMP response to endothelin‐1. Therefore, endothelin‐1 activates guanylate cyclase most probably via formation of nitric oxide, which is released from l‐arginine. 3 The Ca2+ ionophore ionomycin induced a transient rise in cyclic GMP levels, which was also suppressed by preincubation in the presence of either haemoglobin or the l‐arginine analogues l‐canavanine or NG‐monomethyl‐l‐arginine. Therefore, we conclude that ionomycin can activate guanylate cyclase by a mechanism involving nitric oxide formation, similar to that induced by endothelin‐1. 4 The alkaloid veratridine, which activates Na+ channels and also causes influx of Ca2+ induced a transient rise of cyclic GMP levels in the neuronal cell line. This stimulation was blocked by pretreating the cells with l‐canavanine, NG‐monomethyl‐l‐arginine or haemoglobin. 5 Loading the cells with the Ca2+ chelator BAPTA suppresed the cyclic GMP response to application of endothelin‐1, ionomycin, or veratridine. Thus, in the neuronal cell line a rise in cytosolic Ca2+ activity seems to be sufficient to stimulate the nitric oxide forming enzyme which synthesizes the activator of soluble guanylate cyclase.

A novel, specific binding protein assay for quantitation of intracellular inositol 1,3,4,5-tetrakisphosphate (InsP4) using a high-affinity InsP4 receptor from cerebellum

A membrane preparation from porcine cerebellum displays high‐affinity binding sites for [3H]inositol 1,3,4,5‐tetrakisphosphate ([3H]InsP4) with a dissociation constant (K d) of 1.0 nM and a density of 220 fmol/mg protein. Specific binding was maximal in the presence of 25 mM phosphate and at pH 5.0. The receptor site was specific for InsP4, since Ins(1,3,4,5,6)P5 and Ins(1,4,5,6)P4 displaced binding of InsP4 with EC50 values of 0.2 and 0.3 μM, respectively. Ins(1,4,5)P3 and other inositol phosphates were less effective. Using this InsP4 receptor, an assay for measuring tissue content of InsP4 was developed. The detection limit of the assay was 0.1 pmol. In the same tissue samples the amount of Ins(1,4,5)P3 was determined in parallel with a similar assay using a binding protein preparation from beef liver.

Memantine (1-amino-3,5-dimethyladamantane) blocks the serotonin-induced depolarization response in a neuronal cell line

The influence of memantine on several properties of a neuronal cell line was tested. The aim was to get some insight into possible mechanisms of action of this drug which is therapeutically applicable in treatment of spasticity, Parkinsons disease, and cerebral coma. In neuroblastoma X glioma hybrid cells, memantine, at micromolar concentrations, blocked the depolarization induced by iontophoretically applied serotonin (5-hydroxytryptamine, 5-HT). In the hybrid cells, receptors of the 5-HT3 type mediated the depolarization, which was frequently accompanied by a series of action potentials. The inhibition by memantine of the serotonin response occurred fast and was completely reversible, irrespective of whether the cell showed a stable membrane potential or spontaneous action potentials. However, memantine did not alter spontaneous or electrically evoked action potential activity in the hybrid cells, and apparently did not block the underlying ionic conductances. Furthermore memantine did not affect either the cation permeability activated by substance P in the hybrid cells or the K+ channel triggered by bradykinin in a glioma cell line. Thus, memantine appears specifically to suppress the ion channel opened by serotonin in the hybrid cells. The interaction of memantine with serotonin receptors and the associated ion channels reported here, might give an important clue, as to a site of action of memantine in the nervous system.

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 P2u 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).

Substance P and serotonin act synergistically to activate a cation permeability in a neuronal cell line

Both substance P and, to a lesser degree, serotonin activate cation permeability in neuroblastoma x glioma hybrid cells, as determined by measurement of [14C]guanidinium uptake. Serotonin potentiates the action of substance P by shifting the concentration-effect curve of substance P to the left. The EC50 value for the synergistic effect of serotonin was around 0.3 microM. Dopamine and noradrenaline displayed comparable activity, albeit only at 50 and 130 times higher concentrations, respectively. The order of potency of various substance P-analogues was not changed by serotonin, indicating that the specificity of the substance P site on the hybrid cells was not affected by serotonin. Various other neurotransmitters and peptides had no effect on the response of the hybrid cells to substance P. The serotonin receptor interacting with the substance P receptor may be classified as a 5-HT3-receptor since methysergide, cimetidine, and ketanserin were ineffective, but two inhibitors specific for 5-HT3-receptors, ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) and MDL 72222 (1 alpha H,3 alpha,5 alpha H-tropan-3-yl-3,5-dichlorobenzoate), blocked the effect of serotonin at nanomolar concentrations. However, the two serotonin antagonists might also be blocking the ion permeability, since at higher concentrations they fully inhibited the stimulation of guanidinium uptake by substance P or by substance P plus serotonin. The synergism between substance P and serotonin on the hybrid cells offers the opportunity to study the mechanism of interaction of neurotransmitter receptors on a permanent neuronal cell line.

Endothelin Induces a Rise of Inositol 1,4,5-Trisphosphate, Inositol 1,3,4,5-Tetrakisphosphate Levels and of Cytosolic Ca2+ Activity in Neural Cell Lines

The mechanism of action of the vasoconstricting peptide endothelin was investigated in two neural cell lines. In rat glioma cells endothelin‐1 caused a biphasic rise in cytosolic Ca2+ activity. A large peak of 40 s duration was followed by another, however smaller, transient rise of comparable duration. In the absence of extracellular Ca2+ only the first peak was detected. Pretreatment with Ca2+ ionophores suppressed the Ca2+ response to endothelin. At the concentrations used the Ca2+ ionophores primarily deplete internal Ca2+ stores and prevent their refilling. Measurements of 45Ca2+ fluxes corroborate the conclusion that in the glioma cells endothelin induces firstly a release of Ca2+ from internal stores and subsequently a stimulation of Ca2+ entry. In neuronal cells (mouse neuroblastoma × rat glioma hybrid cells), endothelin caused a monophasic rise in cytosolic Ca2+ activity, most likely due to release from internal stores. In the glioma cells the concentrations of both inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate were raised about 2.5‐fold for ca. 90 s after addition of endothelin. In the neuronal cells a shorter, smaller rise in inositololigophosphate concentrations was induced. Thus, endothelin seems to act as a neuropeptide activating phospholipase C and intracellular Ca2+.

Nitric oxide formation caused by Ca2+ release from internal stores in neuronal cell line is enhanced by cyclic AMP

The influence of an elevated level of cyclic AMP on the formation of nitric oxide was investigated in a neuronal cell line (108CC15; NG108-15), in which we had previously shown that nitric oxide mediates the activation of soluble guanylyl cyclase upon stimulation with the hormones bradykinin, endothelin, and serotonin. Maximal amplitude and duration of cyclic GMP response to bradykinin were about 2-fold greater in cells with cyclic AMP levels increased by forskolin pretreatment than in control cells with basal levels of cyclic AMP. Phosphodiesterase inhibitors (isobutylmethylxanthine or M&B 22,948 (zaprinast)) similarly increased the maximal amplitude of the cyclic GMP response to bradykinin, but, in contrast, slowed down the decay phase of the cyclic GMP response to a much greater extent. The cyclic GMP responses to bradykinin were suppressed with the same potency by L-arginine analogues in control and in forskolin-treated cells (IC50 of NG-monomethyl-L-arginine 2 microM, of nitro-L-arginine 0.7 microM). The transient rises of cyclic GMP levels induced by bradykinin and endothelin, which both cause release of Ca2+ from internal stores, were similarly enhanced by forskolin pretreatment. However, the transient cyclic GMP response to serotonin which is due to Ca2+ influx into the neuronal cell line via 5-hydroxytryptamine3 (5-HT3) receptors was not affected by raising the cyclic AMP levels by forskolin pretreatment. Thus, cyclic AMP seems to enhance nitric oxide formation which depends on Ca2+ release from internal stores.

High-affinity inositol 1,3,4,5-tetrakisphosphate receptor from cerebellum: solubilization, partial purification and characterization

Proteins which bind with high affinity Ins 1,3,4,5‐P4 or Ins 1,4,5‐P3 were solubilized from porcine cerebellar membranes. Both binding activities were separated by heparin‐agarose chromatography. The Ins 1,3,4,5‐P4 receptor was partially purified with an approximately 1000‐fold enrichment as compared to the membrane preparation. In the receptor‐enriched preparation the Ins 1,3,4,5‐P413 binding protein had an affinity (K d) for Ins 1,3,4,5‐P4 of 4.6 nM. Ins 1,3,4,5,6‐P5 displaced [3H]Ins 1,3,4,5‐P4binding with a comparable affinity. The Ins 1,3,4,5‐P4binding protein displayed high selectivity for Ins 1,3,4,5‐P4 over other inositolphosphates (IC50 for Ins 1,4,5,6‐P4 150 nM, for Ins‐P6 1 μM and for Ins 1,3,4‐P3 5 μM). Most importantly. Ins 1,4,5‐P3 did not displace [3H]Ins 1,3,4,5‐P4binding at concentrations up to 10μM. Binding of Ins 1,3,4,5‐P4 was maximal in the pH range between 4.5 and 6, was stable with Ca2+ concentration varied from 1 nM to 1 mM, and was suppressed by heparin (IC50 about 2 nM). The high affinity receptor for Ins 1,3,4,5‐P4 reported here, which is distinct from the Ins 1,4,5‐P3 receptor might allow to evaluate the possible functional role of Ins 1,3,4,5‐P4 in the cellular signal transduction.

Mechanisms for activation and subsequent removal of cytosolic Ca2+ in bradykinin-stimulated neuronal and glial cell lines

Mechanisms for activation and for removal of cytosolic Ca2+ after stimulation with bradykinin were investigated in two neural cell lines by measuring cytosolic Ca2+ activity and 45Ca2+ fluxes. In the neuronal (neuroblastoma x glioma hybrid) and in the glial (rat glioma) cell lines, the transient, bradykinin-induced rise in cytosolic Ca2+ activity (determined by fura-2 or indo-1 fluorescence) was blocked by a bradykinin B2 receptor antagonist. Ca2+ ionophores (ionomycin and 4-Br-A23187) caused a comparable transient rise in cytosolic Ca2+ activity. After addition of ionophores, the Ca2+ response to bradykinin was reduced or completely blocked in both cell lines. At the concentrations used, the ionophores primarily depleted intracellular Ca2+ stores and prevented refilling of the stores. Thus, the bradykinin-induced rise of cytosolic Ca2+ activity seems to be mostly due to Ca2+ release from internal stores. In the neuronal but not in the glial cell line, a brief stimulation by bradykinin of 45Ca2+ uptake was followed by a long-lasting inhibition below control values. Thus, in the neuronal cells bradykinin presumably blocks Ca2+ channels by a readily reversible, pertussis toxin-insensitive mechanism. Excess cytosolic Ca2+ of the bradykinin-stimulated cells is mostly not resequestered into the internal Ca2+ pool accessible to bradykinin, but is mainly extruded through the plasma membrane, as indicated by (i) stimulation of 45Ca2+ release by bradykinin, (ii) quick reduction by bradykinin of cellular 45Ca2+ content of cells preequilibrated with 45Ca2+, and (iii) diminution of the ionophore-inducible Ca2+ response after the addition of bradykinin.