Glutamate receptors modulate multiple signaling pathways, several of which involve mitogen-activated protein (MAP) kinases, with subsequent physiological or pathological consequences. Here we report that stimulation of the N-methyl-D-aspartate (NMDA) receptor, using platelet-activating factor (PAF) as a messenger, activates MAP kinases, including c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase, in primary cultures of hippocampal neurons. Activation of the metabotropic glutamate receptor (mGluR) blocks this NMDA-signaling through PAF and MAP kinases, and the resultant cell death. Recombinant PAF-acetylhydrolase degrades PAF generated by NMDA-receptor activation; the hetrazepine BN50730 (an intracellular PAF receptor antagonist) also inhibits both NMDA-stimulated MAP kinases and neuronal cell death. The finding that the NMDA receptor-PAF-MAP kinase signaling pathway is attenuated by mGluR activation highlights the exquisite interplay between glutamate receptors in the decision making process between neuronal survival and death. (+info)
(2/1761) Mechanisms involved in the metabotropic glutamate receptor-enhancement of NMDA-mediated motoneurone responses in frog spinal cord.
1. The metabotropic glutamate receptor (mGluR) agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (10-100 microM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 microM). 2. In the presence of NMDA open channel blockers [Mg2+; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801); 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] and TTX, trans-ACPD significantly potentiated NMDA-induced motoneurone depolarizations, but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA)- or kainate-induced depolarizations. 3. NMDA potentiation was blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) (240 microM), but not by alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (MCCG) (290 microM) or by alpha-methyl-(S)-2-amino-4-phosphonobutyrate (L-MAP4) (250 microM), and was mimicked by 3,5-dihydroxyphenylglycine (DHPG) (30 microM), but not by L(+)-2-amino-4-phosphonobutyrate (L-AP4) (100 microM). Therefore, trans-ACPD's facilitatory effects appear to involve group I mGluRs. 4. Potentiation was prevented by the G-protein decoupling agent pertussis toxin (3-6 ng ml(-1), 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 microM) and N-(2-aminoethyl)-5-isoquinolinesulphonamide HCI (H9) (77 microM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol-12-myristate 13-acetate (5.0 microM) had no effect. 5. Intracellular Ca2+ depletion with thapsigargin (0.1 microM) (which inhibits Ca2+/ATPase), 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) (50 microM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+-free medium all reduced trans-ACPD's effects. 6. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) (100 microM) and chlorpromazine (100 microM) diminished the potentiation. 7. In summary, group I mGluRs selectively facilitate NMDA-depolarization of frog motoneurones via a G-protein, a rise in [Ca2+]i from the presumed generation of phosphoinositides, binding of Ca2+ to calmodulin, and lessening of the Mg2+-produced channel block of the NMDA receptor. (+info)
(3/1761) Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C.
Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypothesized to be a trigger for long-term changes in receptor number and function observed after chronic administration of nicotine at levels similar to those found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicotine. The roles of Ca2+ and protein kinase C (PKC) on desensitization of alpha4beta2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of alpha4beta2 receptors in the presence of Ca2+ developed in a biphasic manner with fast and slow exponential time constants of tauf = 1.4 min (65% relative amplitude) and taus = 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with taurec = 43 min. Recovery was largely eliminated after replacement of external Ca2+ with Ba2+ and slowed by calphostin C (taurec = 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate (taurec = 14 min), a PKC activator, or by cyclosporin A (with taurec = 8 min), a phosphatase inhibitor. alpha4beta2 receptors containing a mutant alpha4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a two-desensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, alpha4beta2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of alpha4 subunits changes the rates governing the transitions from "deep" to "shallow" desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the "permanent" inactivation of alpha4beta2 receptors observed after chronic nicotine treatment. (+info)
(4/1761) Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body.
We have previously shown that stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn -1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body. After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP. Forskolin (0.1 micrometer), an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids. Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body. Our findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers. (+info)
(5/1761) Fas-induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event.
Ligation of the Fas molecule expressed on the surface of a cell initiates multiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis. Our results indicate that cytosolic levels of free (non-complexed) Mg2+ ([Mg2+]i) and Ca2+ ([Ca2+]i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the increases in [Mg2+]i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg2+, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg2+]i is required for apoptosis. Furthermore, we propose that the increases in [Mg2+]i function not only as cofactors for Mg2+-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells. (+info)
(6/1761) Cyclooxygenase-dependent signalling: molecular events and consequences.
Non-steroidal anti-inflammatory drugs (NSAIDs) currently attract large interest. Next to pain relief, NSAIDs have important anti-thrombotic and anti-oncogenic effects. NSAIDs exert their action by inhibition of cyclooxygenase, the enzyme responsible for the production of prostanoids. Prostanoid signal transduction is still poorly understood, but it has become clear that these inflammatory lipids influence cellular physiology at three different levels: (1) activation of a 7 x transmembrane receptor coupled to heterotrimeric G proteins, (2) the inhibition of inflammation by activating corticosteroid-like receptors, (3) participation in receptor protein tyrosine kinase signal transduction. In this review prostanoid signalling at these three different levels will be reviewed and the relevance in (patho)physiological processes will be evaluated. (+info)
(7/1761) Second messenger production in avian medullary nephron segments in response to peptide hormones.
We examined the sites of peptide hormone activation within medullary nephron segments of the house sparrow (Passer domesticus) kidney by measuring rates of hormone-induced generation of cyclic nucleotide second messenger. Thin descending limbs, thick ascending limbs, and collecting ducts had baseline activity of adenylyl cyclase that resulted in cAMP accumulation of 207 +/- 56, 147 +/- 31, and 151 +/- 41 fmol. mm-1. 30 min-1, respectively. In all segments, this activity increased 10- to 20-fold in response to forskolin. Activity of adenylyl cyclase in the thin descending limb was stimulated approximately twofold by parathyroid hormone (PTH) but not by any of the other hormones tested [arginine vasotocin (AVT), glucagon, atrial natriuretic peptide (ANP), or isoproterenol, each at 10(-6) M]. Thick ascending limb was stimulated two- to threefold by both AVT and PTH; however, glucagon and isoproterenol had no effect, and ANP stimulated neither cAMP nor cGMP accumulation. Adenylyl cyclase activity in the collecting duct was stimulated fourfold by AVT but not by the other hormones; likewise, ANP did not stimulate cGMP accumulation in this segment. These data support a tubular action of AVT and PTH in the avian renal medulla. (+info)
(8/1761) Sphingosine 1-phosphate: a prototype of a new class of second messengers.
Sphingosine 1-phosphate (SPP) is an important sphingolipid-derived second messenger in mammalian cells that acts to promote proliferation and to inhibit apoptosis. Various growth factors increase the intracellular concentration of SPP by activating sphingosine kinase, the molecular cloning of which has revealed that it defines a new type of lipid kinase. Cell fate is influenced by the balance between the intracellular concentration of SPP and that of ceramide, a pro-apoptotic sphingolipid metabolite. The observation that a similar "rheostat" is a determinant of cell survival in yeast cells exposed to heat shock indicates that it is an evolutionarily conserved mechanism of stress regulation. SPP also acts extracellularly to inhibit cell motility and to influence cell morphology, effects that appear to be mediated by the G protein-coupled receptor EDG1. These observations indicate that SPP is the prototype of a new class of lipid mediators that exert both intracellular and extracellular actions. (+info)