Ca(2+)-inhibited adenylyl cyclase in cardiac tissue.
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Adenylyl Cyclase Inhibitors↗
Biomedical subjects
G Brooker
Publications and source records attributed to G Brooker.
Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts.
Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-1-phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling pathways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-1-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-1-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-1-phosphate.
3T3 Cells↗
P2U-purinergic receptors on C6-2B rat glioma cells: modulation of cytosolic Ca2+ and cAMP levels by protein kinase C.
The activation of P2-purinergic receptors on C6-2B rat glioma cells caused a transient increase in cytosolic-free Ca2+ concentration ([Ca2+]i) as detected by Fura 2 fluorescence ratio imaging of single cells. These purinergic receptors are of the P2U subtype because UTP and ATP were equipotent and substantially more potent than the P2X- and P2Y-selective agonists alpha,beta-methylene ATP and 2-methylthio ATP, respectively. There was homologous desensitization of the Ca2+ responses between UTP and ATP but no heterologous desensitization between these nucleotides and another Ca(2+)-mobilizing receptor agonist, alpha-thrombin. The UTP-induced peak [Ca2+]i rise was insensitive to chelation of extracellular Ca2+ with EGTA. However, the response was abolished after either depletion of intracellular Ca2+ stores with the microsomal Ca(2+)-ATPase inhibitor thapsigargin or blockade of Ca2+ release from intracellular stores with the muscle relaxant dantrolene. The activation of P2U-purinergic receptors and thrombin receptors increased the formation of total inositol phosphates (IPs) and inhibited cAMP accumulation elicited with either the beta-adrenergic receptor agonist (-)-isoproterenol, or forskolin, a direct activator of adenylyl cyclase. UTP- and alpha-thrombin-induced changes in the levels of IPs, cytosolic Ca2+, and agonist-elicited cAMP accumulation were dramatically inhibited (> 80%) by acute treatment of the cells with the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate but not with the inactive ester 4 alpha-phorbol 12,13-didecanoate. We conclude that in C6-2B cells, the increase in [Ca2+]i after activation of P2U-purinergic receptors is primarily a result of IPs-mediated release of Ca2+ from intracellular stores with secondary influx of Ca2+ by capacitative mechanisms. Also, the inhibition by UTP and alpha-thrombin of agonist-elicited cAMP accumulation is mediated through an increase in [Ca2+]i.
Animals↗
Ca2+ inhibition of beta-adrenergic receptor- and forskolin-stimulated cAMP accumulation in C6-2B rat glioma cells is independent of protein kinase C.
In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.
Animals↗
K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels.
Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.
Animals↗
2-Aminopurine inhibits RNA and protein synthesis and reduces catecholamine desensitization in C6-2B rat glioma cells.
We previously proposed that intracellular cyclic AMP accumulation induces a putative, rapidly turning over protein inhibitory to further hormone activation of adenylate cyclase. In the present study, 2-aminopurine, which has been reported to selectively block c-fos gene expression, was used to test the hypothesis that c-fos protein might be involved in the desensitization to catecholamines was observed in 2-aminopurine-treated C6-2B rat glioma cells. However, we found 2-aminopurine to inhibit, in a concentration-dependent manner, total cellular RNA and protein synthesis in C6-2B, HeLa, Swiss 3T3 and BALB/c cells. mRNA synthesis was also markedly reduced in 2-aminopurine-treated cells. These unexpected findings, while supporting our hypothesis of a protein synthesis-sensitive step in the development of refractoriness, raise concern about the specificity of action of 2-aminopurine to inhibit c-fos induction and thus any cellular process, including desensitization, which might be regulated by c-fos gene expression.
2-Aminopurine↗
Stimulation of high affinity gamma-aminobutyric acidB receptors potentiates the depolarization-induced increase of intraneuronal ionized calcium content in cerebellar granule neurons.
In the treatment of spasticity, the therapeutic cerebrospinal fluid levels of (+/-)-baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, are below 1 microM. However, the mechanism of the therapeutic action of (+/-)-baclofen remains unknown, because, for the most part, the action of (+/-)-baclofen on GABAB receptors requires micromolar concentrations. Using fura-2 fluorescence microscopy, intracellular ionized calcium was measured in cerebellar granule neurons. Stimulation of a high affinity GABAB receptor potentiated by 2-3-fold the rise in intracellular calcium observed after depolarization of the cell with a Krebs Ringer's buffered solution containing 40 mM K+. Both GABA (100 nM) and (+/-)-baclofen (10-100 nM) stimulated this high affinity receptor. The potentiation of the depolarization-induced rise in intracellular calcium by (+/-)-baclofen (100 nM) was completely blocked by the GABAB receptor antagonist CGP 35348 (200 microM). Also, the intracellular calcium response induced by the activation of high affinity GABAB receptors was prevented by dantrolene (10 microM). The cerebellar granule neurons contained calcium-induced calcium release (CICR) stores. Caffeine (3 mM) and ryanodine (100 microM) potentiated the depolarization-induced rise in intracellular calcium, and this response to both drugs was blocked by dantrolene (10 microM). Because dantrolene does not prevent the rise in intracellular calcium after cell depolarization (this calcium originated from the influx of extracellular calcium), (+/-)-baclofen acting via the high affinity GABAB receptor indirectly activates the CICR stores, allowing the influx of extracellular calcium to trigger the release of calcium from these dantrolene-sensitive CICR stores. Thus, this high affinity GABAB receptor might become activated during persistent depolarization caused by pathological states and could be a mechanism to be studied for the therapeutic action of (+/-)-baclofen in spasticity.
Animals↗
Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA.
C6-2B rat glioma cells were stably transfected with substance K receptor cDNA and used to study interactions between cAMP and Ca2+ signaling pathways. Activation of the newly expressed receptors by substance K increased the intracellular free Ca2+ concentration, as monitored by single-cell fura-2 imaging, and markedly inhibited agonist-stimulated cAMP accumulation. Blockade of intracellular Ca2+ mobilization abolished the substance K receptor-mediated inhibition of isoproterenol-induced cAMP production. Phosphodiesterase inhibitors, down-regulation or inhibition of protein kinase C, and pertussis toxin failed to prevent substance K-induced inhibition of agonist-stimulated cAMP accumulation. An increased intracellular Ca2+ concentration caused by either calcium ionophores or activation of endogenous bradykinin receptors was found to markedly reduce cAMP production in wild-type cells. These results demonstrate that elevated intracellular Ca2+ concentration can negatively modulate agonist-stimulated adenylate cyclase activity in C6-2B glioma cells.
1-Methyl-3-isobutylxanthine↗
Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation.
Sphingosine, a metabolite of membrane sphingolipids, regulates proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., N. E. Buckley, K. Gibson. and S. Spiegel. 1990. J. Biol. Chem. 265:76-81). The present study provides new insights into the formation and function of a unique phospholipid, a metabolite of sphingosine, which was unequivocally identified as sphingosine-1-phosphate. The rapid increase in 32P-labeled sphingosine-1-phosphate levels induced by sphingosine was concentration dependent and correlated with its effect on DNA synthesis. Similar to the mitogenic effects of sphingosine, low concentrations of sphingosine-1-phosphate stimulated DNA synthesis and induced pronounced morphological alterations. Both sphingosine and sphingosine-1-phosphate stimulated DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester and sphingosine still elicited similar increases in sphingosine-1-phosphate levels in these cells. Although both sphingosine and sphingosine-1-phosphate acted synergistically with a wide variety of growth factors, there was no additive or synergistic effect in response to a combination of sphingosine and sphingosine-1-phosphate. Using a digital imaging system for measurement of calcium changes, we observed that both sphingosine and sphingosine-1-phosphate are potent calcium-mobilizing agonists in viable 3T3 fibroblasts. The rapid rise in cytosolic free calcium was independent of the presence of calcium in the external medium, indicating that the response is due to the mobilization of calcium from internal store. Our results suggest that sphingosine-1-phosphate may be a component of the intracellular second messenger system that is involved in calcium release and the regulation of cell growth induced by sphingosine.
Animals↗
Regulation of nerve growth factor receptor mRNA content by dexamethasone: in vitro and in vivo studies.
Northern blot hybridization analysis was used to study regulation of nerve growth factor receptor (NGFR) mRNA content by glucocorticoids. Treatment for 6 h with dexamethasone (1 microM) caused a 40% decrease of NGFR mRNA content in PC12 cells and a 60% decrease in C6-2B glioma cells which was time and dose dependent. Dexamethasone (1 microM/kg) administered s.c. for two days to 21-day-old rats, elicits a 60% decrease in NGFR mRNA content in septum. These results suggest that the expression of NGFR gene in the brain could be inhibited by endogenous glucocorticoids. Whether dexamethasone inhibits NGFR gene expression by directly affecting cis-regulatory elements in the promoter regions of the gene remains to be elucidated.
Adrenal Gland Neoplasms↗
Protection by natural and semisynthetic gangliosides from Ca2(+)-dependent neurotoxicity caused by excitatory amino acid (EAA) neurotransmitters.
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Amino Acids↗
Destabilization of ionized Ca2+ homeostasis in excitatory amino acid neurotoxicity: antagonism by glycosphingolipids.
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Amino Acids↗
Gangliosides normalize distorted single-cell intracellular free Ca2+ dynamics after toxic doses of glutamate in cerebellar granule cells.
Glutamate-induced delayed neurotoxicity after abusive and paroxismal activation of its receptors has been proposed to depend upon a sustained increase in intracellular free Ca2+ [( Ca2+]i). To elucidate the temporal and causal relationship between glutamate-induced changes in [Ca2+]i and neuronal death, we simultaneously studied the dynamics of [Ca2+]i changes in single neurons with the acetoxymethyl ester of fura-2 and the cell viability by imaging the nuclear penetration of propidium iodide. The main difference between toxic (50 microM) and nontoxic (5 microM) doses of glutamate is the lack of regulation in [Ca2+]i 20 min after glutamate is removed. This protracted rise in [Ca2+]i in a single cell is correlated with (r = 0.87, P less than 0.01, Spearman's test), and consequently predictive of, the time of appearance of neuronal death, as measured by propidium iodide fluorescence. In addition, the glutamate receptor antagonists dibenzocyclohepteneimine (MK-801) and 3,3-(2-carboxypiperazine-4-yl)propyl 1-phosphate reduce the acute increase of [Ca2+]i induced by glutamate but fail to revert the protracted increase of [Ca2+]i, elicited by toxic doses of glutamate. In contrast, the ganglioside GM1 and the semisynthetic lysoGM1 with N-acetylsphingosine (LIGA-4) and lysoGM1 with N-dichloroacetylsphingosine (LIGA-20) failed to change the immediate rise of [Ca2+]i elicited by glutamate but prevented the protracted increase in [Ca2+]i after toxic doses of glutamate. Voltage-dependent Ca2+ channel blockers (nifedipine, etc.) did not change the initial or protracted responses to glutamate.
Animals↗
Calcium wave evoked by activation of endogenous or exogenously expressed receptors in Xenopus oocytes.
The mRNA encoding the cloned substance K receptor was microinjected into Xenopus laevis oocytes. After expression of the mRNA, Ca2+ was imaged in the oocytes with a digital imaging fluorescence microscopy system using the Ca2(+)-sensitive dyes fura-2 and fluo-3. Application of substance K caused a dose-related wave of Ca2+ mobilization to spread from a focus and to elevate the Ca2+ concentration in the oocyte. Activation of endogenous muscarinic or angiotensin II receptors in noninjected oocytes evoked a similar response. The Ca2+ rise in oocytes induced by substance K was due to internal Ca2+ mobilization and was independent of external Ca2+, since it occurred in Ca2(+)-free medium fortified with 2 mM EGTA. The Ca2+ imaging was well correlated with ion current measurements of voltage-clamped oocytes. Imaging, in addition to detecting the spatial spread of Ca2+ across the cell, was at least as sensitive as voltage clamping and much faster when screening oocytes for the expression of receptor mRNAs that stimulate Ca2+ mobilization. While it is known that fertilization of Xenopus eggs causes a spreading wave of Ca2+ mobilization, we found that activation of either native or newly expressed receptors in oocytes causes a similar change in Ca2+ distribution.
Animals↗
Modulation of glutamate receptors by phencyclidine and glycine in the rat cerebellum: cGMP increase in vivo.
In rats receiving N-methyl-D-aspartate (NMDA) intraventricularly or intracisternally the cerebellar cyclic guanosine monophosphate (cGMP) content increases in a dose-related manner. This response was used to study phencyclidine (PCP) and glycine interactions with the glutamate receptor subtype stimulated by NMDA. The increase of cGMP elicited by NMDA was inhibited by PCP and potentiated by glycine. Moreover, 2-amino-5-phosphonovalerate (APV) abolished the NMDA response. Since the increase in cerebellar cGMP induced by kainate, a synthetic agonist of another glutamate receptor subtype, was not modified by APV, the specificity of its action on NMDA response was confirmed. The increase of cerebellar cGMP content elicited by glycine was inhibited by PCP and APV but not by strychnine. Binding studies failed to demonstrate an apparent competitive interaction between PCP, glycine and NMDA. This suggests that the observed interaction is not of the isosteric type. The present results provide evidence that glycine, in vivo, acting at strychnine-insensitive recognition sites modulates allosterically in a positive manner the function of NMDA-sensitive glutamate receptors.
Amino Acids↗
Regulation of nerve growth factor biosynthesis by beta-adrenergic receptor activation in astrocytoma cells: a potential role of c-Fos protein.
The chain of events that results in increased production of nerve growth factor (NGF) following beta-adrenergic receptor (BAR) stimulation has been investigated in the C6-2B rat astrocytoma cell line. Exposure of these cells to the BAR agonist isoproterenol elicits the following cascade of events: (i) increase of cAMP content; (ii) increase of c-Fos mRNA content; (iii) accumulation of c-Fos protein immunoreactivity in the nucleus; (iv) increase of NGF mRNA content. The increase in c-Fos mRNA and its translation product are early events (15 and 40 min, respectively) and precede the accumulation of NGF mRNA, which peaks at 3 hr. The increase in the two mRNAs appears interrelated because cycloheximide inhibits the accumulation of c-Fos protein and NGF mRNA elicited by isoproterenol. Moreover, the accumulation of nuclear c-Fos protein and NGF mRNA induced by BAR stimulation is reduced by 2-aminopurine, an inhibitor of c-Fos mRNA induction. These data suggest that, in C6-2B astrocytoma cells, the nuclear accumulation of c-Fos protein is required for the induction of NGF mRNA expression by BAR stimulation.
2-Aminopurine↗
Improvements in the automated radioimmunoassay for cAMP or cGMP.
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Autoanalysis↗
Beta adrenergic and prostaglandin receptor activation increases nerve growth factor mRNA content in C6-2B rat astrocytoma cells.
C6-2B rat astrocytoma cells were used to test whether the increase of cellular nerve growth factor (NGF) content and secretion induced by isoproterenol treatment are associated with an increase in the content of mRNA that encodes NGF (NGF mRNA). Incubation of cells with isoproterenol (10 microM) for different time periods produced an increase of NGF mRNA content (3- to 5-fold over control) reaching maximum levels at 3 hr and lasting up to 24 hr. The isoproterenol effect on NGF mRNA was antagonized by the beta adrenergic receptor antagonist I-propranolol (10 microM) but not by phentolamine (10 microM), an alpha adrenergic receptor antagonist. When C6-2B astrocytoma cells were exposed for a short time to isoproterenol (10 microM; 10 or 20 min) followed by a washout period of 3 hr, the NGF mRNA content was increased by about 2-fold. The increase of NGF mRNA was obtained also with 10 microM prostaglandin E1 and this effect was potentiated by 100 microM of 3-isobutyl-1-methylxanthine. Inasmuch as both isoproterenol and prostaglandin E1 increase cyclic AMP content, one can surmise that cyclic AMP is involved in the stimulation of NGF mRNA accumulation. Whether cyclic AMP directly activates NGF gene transcription or activates an intermediate step, however, cannot be assessed by the present experiments.
1-Methyl-3-isobutylxanthine↗