Genetic variability of clutch length in dwarf layers: prospects for poultry breeding.
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Animals↗
Biomedical subjects
A Schobert
Publications and source records attributed to A Schobert.
Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation.
The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300-1000 microM). N-methyl-D-aspartate (20-500 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30-100 microM) and kainate (20 microM). The N-methyl-D-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100 microM) blocked the effect of N-methyl-D-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30 microM) blocked the effect of AMPA and also of glutamate and N-methyl-D-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist D-glutamyl-amino-methanesulfonate (30 microM) blocked the effect of kainate but not of glutamate. Glutamate (1000 microM) did not increase the release of lactate dehydrogenase from astrocytes. Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types-N-methyl-D-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.
Adenosine Triphosphate↗
Differences in the mode of stimulation of cultured rat sympathetic neurons between ATP and UDP.
Postganglionic sympathetic neurons possess at least two excitatory receptors for nucleotides: P2X-purinoceptors and separate receptors for uracil nucleotides. In cultured neurons from rat superior cervical ganglia (SCG), both receptors, when activated, induce release of noradrenaline. Here we describe marked differences between the responses of cultured neurons from rat thoracolumbal paravertebral ganglia to ATP and UDP. ATP elicited release of previously taken up [3H]noradrenaline, induced an inward current, and increased the intra-axonal free calcium level, over the same range of micromolar concentrations. UDP was more potent than ATP in releasing [3H]noradrenaline but induced an inward current only at a concentration of 1 mM and caused much smaller increases in intraaxonal free calcium. The mechanism of action of ATP presumably consists of P2X-purinoceptor activation followed by depolarization, calcium entry through voltage-sensitive channels and exocytosis. The mode of action of UDP is different. It probably activates a G-protein-coupled pyrimidinoceptor. The pyrimidinoceptor then possibly mediates mobilization of intracellular calcium close to the sites of transmitter release and in addition an increase in the calcium sensitivity of the exocytotic apparatus.
Adenosine Triphosphate↗
The influx of Ca2+ and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels.
We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca2+ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca2+]i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker omega-conotoxin (0.1 mumol/l) diminished the release of noradrenaline and the increase of intraterminal Ca2+ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 mumol/l) diminished the release of noradrenaline by 25% and the increase of [Ca2+]i by 39%. The P-type channel blocker omega-agatoxin (0.3 mumol/l) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca2+ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca2+]i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca2+ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
3,4-Diaminopyridine masks the inhibition of noradrenaline release from chick sympathetic neurons via presynaptic alpha 2-adrenoceptors: insights into the role of N- and L-type calcium channels.
We have investigated the participation of the N-type (omega-conotoxin GVIA-sensitive) and L-type (nifedipine-sensitive) calcium channels in the alpha 2-adrenoceptor mediated autoinhibition of the release of [3H]noradrenaline from chick sympathetic neurons in culture. Blockade of 3,4-diaminopyridine-sensitive potassium channels resulted in tetrodotoxin-sensitive and calcium-dependent increase of the release of [3H]noradrenaline evoked by electrical stimulation. Nifedipine attenuated the evoked release under control conditions by 20%, but in the presence of 3,4-diaminopyridine by 51%, while omega-conotoxin decreased the release under control conditions by 87% and in the presence of 3,4-diaminopyridine by only 43%. The L-type calcium channel activator Bay k 8644 increased the evoked release of the transmitter both in the absence and in the presence of 3,4-diaminopyridine. Under control conditions, the alpha 2-adrenoceptor agonist UK 14304 decreased the evoked release by 57% and the alpha 2-adrenoceptor antagonist rauwolscine increased it by 14%. Nifedipine did not prevent this modulation. In the presence of 3,4-diaminopyridine, UK 14304 lost its effect on the release of noradrenaline, but its inhibitory action was restored when nifedipine, but not omega-conotoxin, was added. Changes in the increase of intracellular calcium concentration ([Ca2+]i) evoked by electrical stimulation, measured in the cell processes by microfluorimetry, paralleled the changes in the release of [3H]noradrenaline. Under control conditions, nifedipine attenuated the rise of intracellular calcium by only 16%, while omega-conotoxin did so by 66%. 3,4-Diaminopyridine enhanced the evoked rise of [Ca2+]i; in its presence the rise of intracellular calcium was about equally reduced by nifedipine and omega-conotoxin (by 46 and 36%, respectively). These effects were additive. UK 14304 diminished the peak concentration of [Ca2+]i elicited by the standard electrical stimulation by 31% and rauwolscine antagonised this effect. UK 14304 did not measurably inhibit the stimulation-evoked rise of intraterminal [Ca2+]i in the presence of 3,4-diaminopyridine but it produced an inhibition by 26% if nifedipine had been applied together with 3,4-diaminopyridine. Our observations show that, under control conditions, the stimulated release of [3H]noradrenaline is mainly associated with the opening of N-type channels, while in the presence of 3,4-diaminopyridine the contribution of L-type channels becomes more important. The alpha 2-adrenoceptor stimulation by UK 14304 inhibits the release of [3H]noradrenaline but, in the presence of 3,4-diaminopyridine, the inhibition of release can only be observed if the massive influx through L-type calcium channels is prevented. These data suggest that presynaptic alpha 2-adrenoceptors of chick sympathetic neurons preferentially influence the N-type calcium channels.
4-Aminopyridine↗
P2-purinoceptors on postganglionic sympathetic neurones.
1. Postganglionic sympathetic neurones possess both excitatory and inhibitory P2-purinoceptors. 2. The mechanisms of action of excitatory P2-purinoceptors have recently been studied on cultured sympathetic neurones of the rat. The receptors mediate fast increases in intracellular Ca2+ levels and a release of noradrenaline. They are likely to belong to the neuronal types of P2X-purinoceptors and to be located on the sympathetic nerve cell bodies or their dendrites. 3. Inhibitory P2-purinoceptors have been shown to operate at sympathetic axon terminals in isolated tissues. Adenine nucleotides decreased the stimulation-evoked release of noradrenaline by activation of these receptors. The receptors are likely to belong to the group of G-protein-coupled P2Y-purinoceptors. They mediate a negative feedback in which co-transmitter ATP inhibits subsequent sympathetic transmitter release.
Action Potentials↗
Cultured chick sympathetic neurons: modulation of electrically evoked noradrenaline release by P2-purinoceptors.
The present study investigates the pharmacological profile of P2-purinoceptors modulating noradrenaline release from cultured chick sympathetic neurons. ATP (30 microM-3 mM) and 2-methylthio-ATP (3-100 microM), but not alpha, beta-methylene-ATP (up to 100 microM), caused a significant facilitation of electrically evoked [3H]-noradrenaline release when added 2 min before depolarization. The facilitation declined with time of exposure suggesting receptor desensitization. The facilitatory effect was markedly diminished by the P2-purinoceptor antagonists reactive blue 2 (3 microM) and suramin (300 microM), but not changed by mecamylamine (10 microM), a nicotinic receptor antagonist. At 1 mM and higher concentrations, ATP added for 12 min, inhibited noradrenaline release; release was virtually abolished by 6 mM ATP. The inhibitory effect of ATP was slightly diminished by suramin but not affected by reactive blue 2. Electrically evoked [3H]-noradrenaline release remained unaffected in the presence of the adenosine (P1)-receptor agonists R(-)N6-(2-phenylisopropyl)adenosine (R-PIA), 2-[p-(2-carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine (NECA), and N6-2-(4-aminophenyl)ethyladenosine (APNEA), used up to 1 microM. The present results confirm the existence of two P2-purinoceptors affecting noradrenaline release: 1) a facilitatory receptor which is activated by 2-methylthio-ATP as well as ATP, and blocked by suramin as well as reactive blue 2, and 2) an inhibitory receptor which is activated by ATP, only slightly affected by suramin but not at all by reactive blue 2 and does not belong to the established P2-purinoceptor subtypes.
Adenosine Triphosphate↗
Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists.
The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [3H]-noradrenaline was investigated. Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [3H]-noradrenaline (0.05 microM), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 microM-3 mM), 2-methylthio-ATP (3-100 microM), as well as DMPP (10 and 100 microM) induced a significant overflow of tritium. The EC50-value of ATP was 20 microM. Both the ATP-induced and the DMPP-induced tritium overflow was Ca(2+)-dependent and sensitive to tetrodotoxin (0.3 microM) and omega-conotoxin (0.1 microM); in addition, it was inhibited by the alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 microM). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P2-purinoceptor antagonists suramin (300 microM) and reactive blue 2 (3 microM); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 microM) and markedly inhibited by hexamethonium (100 microM). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [3H]-choline. The results suggest that ATP at mumolar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.
Adenine Nucleotides↗
Differences of the electrical and nicotinic receptor stimulation-evoked liberation of norepinephrine from chicken sympathetic neurons in culture: possible involvement of different pools of the transmitter.
We studied the release of [3H]norepinephrine from chicken sympathetic neurons in culture evoked by nicotinic and electrical stimulation with an intention to establish functional identity or nonidentity of the two stimuli in investigations of neurotransmitter release. Nicotinic stimulation evoked extracellular calcium dependent release of [3H]norepinephrine and the rise of intracellular calcium concentration. The release was completely blocked by nicotinic antagonists hexamethonium (100 mumol/l) and mecamylamine (10 mumol/l), and decreased by tetrodotoxin (0.3 mumol/l) and omega-conotoxin (0.1 mumol/l) to 17% and 27%, resp. The intracellular calcium response was decreased by nicotinic antagonists and tetrodotoxin, but not changed by omega-conotoxin. The electrical stimulation-evoked release was blocked by both tetrodotoxin and omega-conotoxin, and decreased by previous electrical, but not nicotinic, stimulation. The differential sensitivity to omega-conotoxin and tetrodotoxin,and the inability of nicotinic stimulation to decrease the liberation by following electrical stimulation may suggest the mobilization of different pools of the transmitter.
Action Potentials↗
Presynaptic nicotinic receptors stimulate increases in intraterminal calcium of chick sympathetic neurons in culture.
Stimulation of chick sympathetic neurons in culture by the cholinergic agonists acetylcholine, nicotine, and 1,1-dimethyl-4-phenylpiperazinium (all at 10-1,000 mumol/L) induced concentration-dependent increases of free calcium levels measured by fura 2 fluorescence in neuronal processes. The response evoked by acetylcholine had both nicotinic and muscarinic components, whereas that induced by 1,1-dimethyl-4-phenylpiperazinium was purely nicotinic. Tetrodotoxin (0.3 mumol/L) blocked completely the increase of intraterminal free calcium level evoked by electrical stimulation. On the other hand, stimulation with 1,1-dimethyl-4-phenylpiperazinium still evoked 20-25% of the control response in the presence of tetrodotoxin. The concentration-response relationship of 1,1-dimethyl-4-phenylpiperazinium stimulation did not differ in the absence and in the presence of tetrodotoxin. The nicotinic antagonists d-tubocurarine (10 mumol/L) and mecamylamine (10 mumol/L), but not alpha-bungarotoxin (125 nmol/L), prevented the increase of intraterminal free calcium level evoked by 1,1-dimethyl-4-phenylpiperazinium (100 mumol/L) in the presence of tetrodotoxin. These observations indicate the presence of nicotinic receptors on neuronal processes that increase the intraterminal concentration of free calcium and probably modulate transmitter release. Their pharmacological properties are similar to those of nicotinic receptors located on neuronal cell bodies.
Acetylcholine↗
Presynaptic alpha 2-adrenoceptors inhibit calcium influx in terminals of chicken sympathetic neurons and noradrenaline release evoked by nicotinic stimulation.
The changes of intracellular calcium concentration evoked by stimulation of nicotinic receptors and its modulation by alpha 2-adrenoceptors were investigated in chicken sympathetic neurons grown in culture. Stimulation of nicotinic receptors increased the intracellular calcium both in the area of cell bodies and processes. In normal extracellular calcium (1.3 mmol/l), the specific alpha 2-adrenoreceptor agonist UK 14,304 (10 mumol/l) diminished the response in cell bodies but not in processes. The same treatment in medium with reduced calcium (0.13 mmol/l) resulted in the decrease of the response evoked by nicotinic stimulation in the area of processes but not in the cell bodies. The effect of UK 14,304 on evoked noradrenaline release paralleled its influence on intracellular calcium in processes. These data indicate that nicotine stimulation-induced influx of calcium is inhibited by alpha 2-adrenoceptor stimulation both in cell bodies and in processes. It is concluded that the nicotinic stimulation-evoked release of noradrenaline is triggered by influx of calcium through alpha 2-adrenoreceptor sensitive as well as insensitive pathways.
Adrenergic alpha-2 Receptor Agonists↗
P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture.
ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 microM), a P2Y-selective purinoceptor agonist, increased electrically evoked release of [3H]noradrenaline from chick sympathetic neurons. The P2X-selective purinoceptor agonist alpha,beta-methylene-ATP (30 microM) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P1) receptor antagonist 8-(p-sulfophenyl)-theophylline (8-PST; 100 microM), but completely prevented by the non-selective P2 antagonist suramin (300 microM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P2Y purinoceptor, whereas inhibition is caused by a P2 purinoceptor which needs further subtype characterization.
Adenosine Triphosphate↗
Modulation of electrically evoked [3H]-noradrenaline release from cultured chick sympathetic neurons.
In the present study we attempted a comprehensive characterization of modulation of noradrenaline release from chick sympathetic neurons. To this purpose sympathetic neurons derived from chick lumbosacral paravertebral ganglia and kept in culture for 7 days were loaded with 0.05 mumol/l [3H]-noradrenaline and subjected to electrical field stimulation (36 pulses/3 Hz). Since the released transmitter was partially recaptured, superfusion was usually performed in the presence of (+)-oxaprotiline, an inhibitor of noradrenaline re-uptake. [3H]-Noradrenaline was released in a manner which was dependent on extracellular Ca2+ and sensitive to tetrodotoxin (TTX). omega-Conotoxin (omega-CTX; 100 nmol/l) abolished [3H]-noradrenaline release indicating that influx through omega-CTX-sensitive Ca(2+)-channels was essential for transmitter release. 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2- (trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester ((+/-) Bay K 8644) and 4-(4-benzofurazanyl)-1,4-dihydro-2,6- dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester ((+)-202-791), agonists at L-type voltage sensitive Ca(2+)-channels (VSCCs), increased noradrenaline release and induced, in addition, an overflow of tritium which was Ca(2+)-dependent and prevented by the presence of TTX. The L-type VSCC antagonists (-)-202-791 and (+)-4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5- pyridinedicar boxylic acid methyl, isopropyl ester) ((+)-PN 200-110) diminished [3H]-noradrenaline release. These data suggest that L-type VSCCs, probably located on the cell body of the neuron, play an additional role in modulation of release.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic alpha-Agonists↗
Rat microglial interleukin-3.
Interleukin-3 (IL-3, multi-CSF) is a growth factor for a variety of hematopoietic progenitor cells. Recently, microglial cells, the resident macrophages of the central nervous system (CNS) have been shown to proliferate in the presence of IL-3 both in vivo and in culture. Data obtained from cultured astrocytes gave rise to the hypothesis that astrocytes synthesize the microglial growth factor. This is the first report identifying rat microglial cells themselves as a source of IL-3. Culture media conditioned by isolated microglia enhanced microglial proliferation above fresh media controls. IL-3 polypeptide was detected in both conditioned media (CM) and in microglial cells by Western blotting and immunoprecipitation. Furthermore, anti-IL-3 antibodies were able to inhibit microglial proliferation induced by conditioned media. mRNAIL-3 was present in single microglial cells as revealed by in situ hybridization. Total RNA prepared from purified microglia yielded a single PCR amplification product. Identity of the PCR product was confirmed by Southern blot hybridization using a cDNAIL-3 probe and by DNA sequencing. Expression of mRNAIL-3 was observed in both absence and presence of lipopolysaccharide, a bacterial endotoxin, that commonly induces expression of inflammatory cytokines and inhibits microglial proliferation. It is concluded that IL-3 expression in ensuring the recruitment of enhanced numbers of immunocompetent cells at sites of lesion. In the light of weak immune reactions in the brain, it is hypothesized that the expression of a characteristic T cell feature in monocyte-derived microglia may be a partial compensation of T cell functions in brain lesions.
Animals↗
The phorbol ester TPA potentiates cholera toxin- and isoproterenol-stimulated cyclic AMP-synthesis in primary astrocyte cultures.
Cellular responses to changes in the extracellular environment are mediated by intracellular signaling systems. One of the most extensively studied systems is adenylate cyclase which generates the second messenger molecule cAMP. Another one is the phosphatidylinositol (PI) second messenger system giving rise to IP3 and diacylglycerol, the latter stimulating protein kinase C. Recently, a third potential signaling system has attracted increased scientific attention: the phospholipase A2 system which generates arachidonic acid. This substance may be used for eicosanoid synthesis or serve as a second messenger molecule. The present report gives more evidence about mechanisms how these signaling pathways interact in cultured astrocytes. Substances commonly used for stimulation of arachidonic acid release and prostaglandin synthesis in these cultures (A23187, TPA) had no influence on intracellular cAMP levels. Pertussis toxin that had previously been shown to inhibit prostaglandin synthesis, had no influence on cAMP levels either. Cholera toxin, however, raised intracellular cAMP significantly, although much less than the beta-adrenoceptor agonist isoproterenol. Cholera toxin also caused a marked change in astroglial morphology even at reduced concentrations (1-10 ng/ml). A23187 used in combination with Ctx had a moderate stimulatory effect on cAMP synthesis. In contrast, in the presence of Ctx, the PKC-activating phorbol ester TPA synergistically stimulated cAMP production, raising cAMP levels as high as isoproterenol-stimulated levels. The TPA effect was concentration-dependent. It was also dependent on an intact PKC since preincubation of cells with the phorbol ester completely abolished the synergistic effect. The synergistic effect of the phorbol ester was also observed at subthreshold concentrations of isoproterenol. The data reveal that the sole activation of most Gs molecules is a necessary but not sufficient prerequisite to achieve maximal adenylate cyclase activity. The fine-tuning of this activity apparently occurs at the catalytic subunit which is under the (partial) control of phosphorylation by PKC.
Adrenergic beta-Agonists↗
Co-release of noradrenaline and ATP from cultured sympathetic neurons.
The vesicles of postganglionic sympathetic axons store both noradrenaline and ATP. The theory of noradrenaline-ATP co-transmission implies that both compounds are released by nerve action potentials and elicit postjunctional effects. Many properties of postjunctional responses support the theory. However, neural release of ATP has been difficult to detect biochemically: by far the major part of the overflow of ATP from intact tissues upon sympathetic nerve stimulation comes from non-neural elements, especially smooth muscle and endothelial cells. Here we describe a parallel electrically evoked overflow of [3H]noradrenaline and endogenous ATP from cultured chick sympathetic neurons. The overflow was abolished by tetrodotoxin, omega-conotoxin and withdrawal of Ca2+, was increased by tetraethylammonium and 4-aminopyridine, and was not changed by prazosin or suramin. The results demonstrate directly the action potential-evoked, Ca(2+)-dependent and presumably vesicular and exocytotic release of ATP from postganglionic sympathetic neurons. They support the co-transmitter theory and suggest that cultured sympathetic neurons are a preparation in which noradrenaline-ATP co-release can be examined free from postjunctional components.
4-Aminopyridine↗
Secretogranin II is synthesized and secreted in astrocyte cultures.
Astrocyte cultures from rat brain were analyzed for their ability to synthesize and secrete secretogranin II (chromogranin C). Northern blot analysis of polyA-selected RNA established the presence of secretogranin II mRNA in these cells. By radioimmunoassay, 11.6 fmol/10(6) astrocytes of secretogranin II was found in these cells. About twice the amount was released into the medium within 3 days. Secretogranin II within the astrocytes was practically unprocessed, as shown by HPLC. These results establish for the first time that astrocytes in vitro synthesize and secrete a protein of the acidic chromogranin family.
Animals↗
Functionally diverse purinergic P2Y-receptors mediate prostanoid synthesis in cultured rat astrocytes: the role of ATP-induced phosphatidyl-inositol breakdown.
Cultured rat astrocytes possess purinergic P2Y-receptors. Stimulation of these receptors with ATP (10(-3) M) results in increased phosphatidylinositol biphosphate (PIP2)-breakdown and prostanoid formation. We have investigated the relevance of the PIP2-pathway in prostanoid synthesis. The intracellular Ca(2+)-mobilizing agent thapsigargin (TG) (10(-6) M) and the diacylglycerol (DAG)-mimetic tetradecaoylphorbol acetate (TPA) (10(-8)-10(-6) M) both stimulate prostaglandin D2 production. ATP-induced prostanoid formation can be mimicked by combined addition of TG and TPA, suggesting the importance of the second messengers IP3 and DAG, generated during P2Y-receptor mediated PIP2-breakdown. Inhibition of ATP-induced PIP2-hydrolysis by TPA (IC50 about 5 x 10(-8) M) or by 10(-4) M neomycine, however, does not affect astroglial prostanoid synthesis, showing that P2Y-receptor mediated prostanoid formation may occur also in the absence of PIP2-hydrolysis. These findings suggest that additional postreceptor mechanisms exist in the signal transduction chain of ATP-induced astroglial prostanoid synthesis. A possible involvement of phospholipase A2 and/or of Ca(2+)-channels, directly coupled to P2Y-receptors is proposed.
Adenosine Triphosphate↗