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C Sumners

Publications and source records attributed to C Sumners.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors.

Neurons cultured from neonatal rat hypothalamus and brainstem contain many angiotensin II (Ang II) type 2 (AT2) receptors, and we previously determined that activation of these sites elicited a stimulation of serine/threonine phosphatase 2A (PP2A). Here, we have investigated the effects of Ang II on neuronal mitogen-activated protein (MAP) kinases, potential targets for PP2A. Using in-gel kinase assays and immunoprecipitation analyses we have shown that Ang II (10 nM-1 microM) elicits significant increases in p44(MAPK) (Erk1) and p42(MAPK) (Erk2) activities in cultured neurons, mediated via Ang II type 1 (AT1) receptors. This stimulatory effect of Ang II on Erk1 and Erk2 activities was potentiated by blockade of AT2 receptors with (S)-1-[4-(dimethylamino)-3-methylphenyl]methyl-5-(diphenylacetyl)- 4, 5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid (PD 123319, 1 microM). Furthermore, the AT2 receptor agonist N-alpha-nicotinoyl-Tyr-Lys-(N-alphaCBZ-Arg)-His-Pro-Ile-OH (CGP42112A) (10-50 nM) caused significant decreases in neuronal Erk1 and Erk2 activities, which were abolished by PD 123319 (1 microM) and by the PP2A inhibitor okadaic acid (3 nM). This indicates that AT1 and AT2 receptors have opposite actions on Erk1 and Erk2 activities in neonatal neurons. Since MAP kinases are involved in the regulation of growth/differentiation and apoptosis, our data may provide an intracellular basis for modulatory effects of Ang II receptors on these processes.

Angiotensin Receptor Antagonists↗
PubMed1996

Angiotensin II stimulates protein phosphatase 2A activity in cultured neuronal cells via type 2 receptors in a pertussis toxin sensitive fashion.

Recent studies have suggested a role for an inhibitory G protein (Gi) and protein phosphatase 2A (PP2A) in the angiotensin II (Ang II) type 2 (AT2) receptor mediated stimulation of neuronal K+ currents. In the present study we have directly analyzed the effects of Ang II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Ang II elicited time (30 min-24 h)- and concentration (10 nM -1 microM)-dependent increases in PP2A activity in these cells. This effect of Ang II involved AT2 receptors, since it was inhibited by the AT2 receptor selective ligand PD123319 (1 microM), but not by the Ang II type 1 receptor antagonist losartan (1 microM). Furthermore, the stimulatory effects of Ang II on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (PTX) (200 ng/ml; 24 h) indicating the involvement of an inhibitory G-protein; and by cycloheximide (CHX) (1 microgram/ml; 30 min) indicating a requirement for protein synthesis. These effects of Ang II appear to be via activation of PP2A, since Western Blot analyses revealed no effects of this peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a key component of the intracellular pathways coupled to neuronal AT2 receptors.

Angiotensin II↗
PubMed1996

Angiotensin II regulation of intracellular calcium in astroglia cultured from rat hypothalamus and brainstem.

This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1-100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura-2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl2), the Ang II-stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady-state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 microM) inhibited the Ang II-stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 microM) did not inhibit the Ang II-stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II-stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca(2+)-free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 microM), cyclopiazonic acid (10 microM), and ryanodine (10 microM), but not caffeine (1-10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 microM) and nifedipine (10 microM); in contrast, gadolinium (100 microM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.

Angiotensin II↗
PubMed1996

Protein kinase C modulates natriuretic peptide receptors in astroglial cultures from rat brain.

We determined previously that astroglia cultured from newborn rat brain contain both guanylyl cyclase-coupled and atrial natriuretic peptide (ANP)-C natriuretic peptide receptors. Here, we investigated the effects of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) on these receptor subtypes in cultured astroglia to understand the intracellular processes involved in the modulation of natriuretic peptide receptors in these cells. PMA (10 nM to 1 microM; 15 min to 24 h) treatment elicited a time- and concentration-dependent decrease in the numbers of 125I-labeled ANP specific binding sites, which was inhibited by the PKC antagonist staurosporine (500 nM). Furthermore, PMA (100 nM, 2 or 24 h) treatment elicited a significant decrease in the specific binding of 125I-des-Cys-Cys-ANP, an ANP-C receptor selective ligand. PMA (10 nM to 1 microM; 30 min) treatment also significantly decreased ANP (100 nM)-stimulated guanosine 3', 5'-cyclic monophosphate levels in cultured astroglia, an effect unmodified by phosphodiesterase inhibition. These data indicate that PKC modulates both guanylyl cyclase-coupled and ANP-C natriuretic peptide receptors in cultured astroglia.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms.

Angiotensin II (ANG II) elicits an ANG II type 1 (AT1) receptor-mediated decrease in voltage-dependent K+ current (Ik) and an increase in voltage-dependent Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem. Modulation of these currents by ANG II involves intracellular messengers that result from an AT1 receptor-mediated stimulation of phosphoinositide hydrolysis. For example, the effects of ANG II on IK and ICa were abolished by phospholipase C antagonists. The reduction in IK produced by ANG II was attenuated by either protein kinase C (PKC) antagonists or by chelation of intracellular Ca2+. By contrast, PKC antagonism abolished the stimulatory effect of ANG II on ICa. Superfusion of the PKC activator phorbol 12-myristate 13-acetate produced effects on IK and ICa similar to those observed after ANG II. Furthermore, intracellular application of inositol 1,4,5-trisphosphate (IP3) elicited a significant reduction in IK. This suggests that the AT1 receptor-mediated changes in neuronal K+ and Ca2+ currents involve PKC (both IK and ICa) and IP3 and/or intracellular Ca2+ (IK).

Angiotensin II↗
PubMed1996

Angiotensin II type 2 receptor-mediated regulation of rat neuronal K+ channels.

We have previously shown that angiotensin II (Ang II), via AT2 receptors, increases whole-cell K+ current in cultured rat hypothalamus and brain stern neurons. We have now investigated the AT2 receptor-mediated effects of Ang II on the activity of single delayed rectifier K+ channels in cell-attached membrane patches. In control recordings (bath, 5.4 mmol/L K+; pipette, 140 mmol/L K+), two voltage-dependent channels were recorded with conductances of 34 +/- 4 and 56 +/- 6 pS, respectively (n = 6). When patches were excised, the channels reversed near a membrane potential expected for a K+ channel. In cell-attached patches (-40 mV), Ang II (100 nmol/L) increased open probability of the 56-pS K+ channel from 0.03 +/- 0.01 to 0.21 +/- 0.05 (n = 3). The selective AT2 receptor antagonist PD 123319 (1 mumol/L) but not the AT1 receptor antagonist losartan (1 mumol/L) blocked the actions of Ang II (n = 3). The selective AT2 receptor agonist CGP 42112 (100 nmol/L) produced similar effects to Ang II. Kinetic analysis of the Ang II effect showed that open-time histograms were best fit by two exponential functions. Ang II increased both open-time constants relative to control (control, tau 1 = 0.9 +/- 0.1 milliseconds, tau 2 = 2.3 +/- 0.3 milliseconds; Ang II, tau 1 = 3.1 +/- 0.4 milliseconds, tau 2 = 12.1 +/- 2.4 milliseconds), and PD 123319 blocked this effect (n = 3). The closed-time histogram was not affected by Ang II PD 123319, or losartan. These results suggest that activation of AT2 receptors modulates rat hypothalamus and brain stern neuronal whole-cell K+ current by increasing the open probability of a 56-pS K+ channel.

Angiotensin II↗
PubMed1995

Regulation of secretogranin II mRNA in rat neuronal cultures.

The regulation of SgII mRNA expression was investigated in primary cultures of neurons prepared from the hypothalamus and brainstem of 1-day-old rats. The administration of forskolin (FSK) resulted in a time- and dose-dependent increase in SgII mRNA expression, a 9-fold effect within 6 h being achieved with 10 microM FSK, which maximally increased cellular cAMP levels. SgII mRNA levels remained elevated for 24 h. Activation of protein kinase C with 100 nM phorbol 12-myristate 13-acetate (PMA) also increased SgII mRNA expression, although induction with PMA was slower and more moderate (3.8-fold above control after 24 h). Neither 10 microM 1,9-dideoxyforskolin nor 100 nM 4 alpha-phorbol 12,13-didecanoate, inactive analogues of FSK and PMA respectively, had an effect on SgII mRNA. Depolarization of neuronal cultures with 50 mM KCl had a small and variable effect on SgII mRNA levels (1.8-fold above control) in neuronal cultures and did not influence induction with FSK. To investigate whether neuron-like regulation of SgII mRNA expression could be reproduced in PC12 cells, PC12 cells were treated with 100 nM nerve growth factor (NGF) for 7 days prior to challenge with FSK or PMA. Whereas NGF alone modestly increased SgII mRNA expression in PC12 cells (1.8-fold above control), it did not uncover a stimulatory effect of FSK or PMA. These studies indicate that SgII mRNA expression is enhanced by an increase in cellular cAMP and activation of protein kinase C in primary cultures of neurons and emphasize that SgII mRNA is regulated in a cell-specific manner.

Animals↗
PubMed1995

Angiotensin II type 2 receptor-mediated stimulation of protein phosphatase 2A in rat hypothalamic/brainstem neuronal cocultures.

Recent studies have suggested a role for an inhibitory guanine nucleotide binding (Gi) protein and protein (serine/threonine) phosphatase 2A (PP2A) in the angiotensin II type 2 (AT2) receptor-mediated stimulation of neuronal K+ currents. In the present study we have directly analyzed the effects of angiotensin II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Angiotensin II elicited time (30 min-24 h)- and concentration (10 nM-1 microM)-dependent increases in PP2A activity in these cells, an effect mimicked by the AT2 receptor ligand CGP-42112A. These effects of angiotensin II and CGP-42112A involve AT2 receptors, because they were inhibited by the AT2 receptor-selective ligand PD 123,319 (1 microM) but not by the angiotensin II type 1 receptor antagonist losartan (1 microM). Furthermore, the stimulatory effects of angiotensin II and CGP-42112A on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (200 ng/ml; 24 h), indicating the involvement of a Gi protein. These effects of angiotensin II and CGP-42112A appear to be via activation of PP2A, and western blot analyses revealed no effects of either peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a cellular target modified following neuronal AT2 receptor activation.

Angiotensin II↗
PubMed1995

Modulation of the delayed rectifier K+ current in neurons by an angiotensin II type 2 receptor fragment.

Angiotensin II (ANG II) stimulates the delayed rectifier K+ current (IK) in neurons cultured from rat hypothalamus and brain stem via AT2 receptors, and this effect involves activation of a Gi protein and protein phosphatase 2A (PP2A). However, there was no evidence that the AT2 receptor involved in this response was the same as the recently cloned AT2 receptor. In the present study, intracellular injection of a 22-amino acid peptide (PEP-22) corresponding to the putative third intracellular loop of the cloned AT2 receptor elicited an increase in IK in cultured neurons that was similar to the effect produced by ANG II. Furthermore, this effect of PEP-22 was abolished by pertussis toxin (200 ng/ml, 24 h) pretreatment and also by superfusion of the PP2A inhibitor okadaic acid (10 nM), suggesting the involvement of Gi protein and PP2A, respectively. Intracellular injection of a random peptide or normal pipette solution did not affect neuronal IK. This is direct evidence to link the cloned AT2 receptor to a defined response elicited by ANG II.

Amino Acid Sequence↗
PubMed1995

Angiotensin II decreases inducible nitric oxide synthase expression in rat astroglial cultures.

Consistent with stimulation of expression of an inducible form of nitric oxide synthase (iNOS), exposure of rat astroglial cultures to lipopolysaccharide (LPS) caused a time-dependent increase in the accumulation of nitrite in the culture media. Addition of the peptide angiotensin II (ANG II) with LPS decreased subsequent formation of nitrite in a concentration-dependent manner (concentration inhibiting 50% of maximal response approximately 1 nM). The ANG II effect could be blocked by the ANG II type 1 (AT1 receptor antagonist losartan but not by the ANG II type 2 (AT2) receptor antagonist PD-123177. ANG II had no effect on nitrite formation stimulated by a combination of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma). A brief 10-min exposure to ANG II was sufficient to cause an approximately 30% inhibition of the LPS response, with maximal inhibition of approximately 65% after 3 h, and occurred only when ANG II was added during the iNOS induction phase. Consistent with partial inhibition of LPS-stimulated expression of iNOS, ANG II reduced the levels of both iNOS mRNA and iNOS protein. These results demonstrate that ANG II can decrease LPS-stimulated NO production in astroglia by inhibiting induction of iNOS expression.

Amino Acid Oxidoreductases↗
PubMed1995

Angiotensin II increases glucose uptake and glucose transporter-1 mRNA levels in astroglia.

In this study, we have investigated the effects of angiotensin II (ANG II) on glucose uptake into astroglia cultured from adult rat hypothalamus and brain stem. ANG II (30 min to 4 h; 10(-9) to 10(-6) M) stimulated time- and concentration-dependent increases in the uptake of 2-deoxy-D-[3H]glucose into cultured astroglia. This effect of ANG II (10(-7) M) is via AT1 receptors and protein kinase C (PKC), since it was inhibited by losartan (10(-6) M) and staurosporine (10(-6) M), respectively. Furthermore, this ANG II action was inhibited by both cycloheximide (1 microgram/ml) and actinomycin D (10(-6) M), indicating that synthesis of new glucose transporters is involved. This was confirmed by the finding that ANG II (30 min to 4 h; 10(-9) to 10(-5) M) stimulated time- and concentration-dependent increases in the steady-state levels of glucose transporter-1 (GLUT-1) mRNA in these cultures. In addition, the increase in steady-state levels of GLUT-1 mRNA elicited by ANG II was mediated by AT1 receptors and PKC. These data suggest that ANG II stimulates glucose uptake into cultured astroglia via a pathway that involves AT1 receptors, PKC, and increased steady-state levels of GLUT-1 mRNA.

Angiotensin II↗
PubMed1994

Peptide receptors in astroglia: focus on angiotensin II and atrial natriuretic peptide.

Astroglial cells derived from the mammalian central nervous system contain a wide variety of peptide receptors, including specific sites for angiotensin II (AII) and atrial natriuretic peptide (ANP). The AII receptors present in these cells are primarily of the AT1 subtype. The ANP receptors present in these cells consist of a mix of ANP-A and ANP-B sites ("biological receptors") and also ANP-C sites ("clearance receptors"). Available evidence indicates that activation of AII receptors results in a stimulation of astroglial proliferation, whereas ANP has an antiproliferative effect in these cells. Intracellular pathways which may mediate these effects of AII and ANP on cell proliferation are discussed, including the presentation of novel data on the activation of protein kinase C and of glucose uptake by AII. We also consider the possibility that the opposing actions of AII and ANP on astroglial proliferation may represent another facet of the mutual antagonism between these two peptides, which has been observed throughout mammalian systems.

Angiotensin II↗
PubMed1994

Receptor-mediated effects of angiotensin II on neurons.

Aside from its well-known and numerous actions at peripheral tissues, the octapeptide angiotensin II (ANG II) elicits specific receptor-mediated effects within the central nervous system. In this review we focus on the receptor-mediated actions of ANG II on neurons. The distribution of ANG II receptors in the brain and physiological, electrophysiological, and cellular effects mediated by these receptors are discussed. This is extended to a review of the characteristics of ANG II receptor subtypes on cultured neurons and the cellular and genomic actions mediated by these receptors. Finally, we develop this information into speculative models for the cellular effects mediated by each ANG II receptor subtype in neurons.

Angiotensin II↗
PubMed1994

Regulation of angiotensin II type 1 receptor mRNA in neuronal cultures of normotensive and spontaneously hypertensive rat brains by phorbol esters and forskolin.

Neuronal cells in primary culture from the brains of normotensive, Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats express angiotensin II type 1 (AT1) receptors. Treatment of WKY rat brain cultures with a phorbol ester, phorbol 12-myristate 13-acetate (PMA), causes a time- and dose-dependent increase in the levels of an approximately 2.3-kb AT1 receptor mRNA transcript. A maximal stimulation of 4.5-fold in the AT1 receptor mRNA transcript level is observed with 200 nM PMA in 4 h and is blocked by 1 microM staurosporine. Forskolin also increases the AT1 receptor mRNA levels in WKY rat brain neurons in a time- and dose-dependent manner, and a 4.5-fold stimulation is achieved with 50 microM forskolin in 4 h. The stimulatory effects of both PMA and forskolin are completely abolished by coincubation of neuronal cultures with 1 microM actinomycin D. In addition, nuclear run-on assay indicated an increase in the transcription of AT1 receptor mRNA in WKY rat brain neurons treated with either PMA or forskolin. Both PMA and forskolin also stimulate levels of AT1 receptor mRNA in neuronal cultures from brain of the SH rat. The degree of stimulation in these cultures is comparable to that in WKY rat brain neurons. These observations show that although the basal AT1 receptor gene expression is significantly higher in SH rat brain neurons compared with WKY rat brain neurons, the protein kinase C- and protein kinase A-responsive stimulation is not altered. These data suggest a possible involvement of protein kinase C and protein kinase A response elements in AT1 receptor gene expression.

Animals↗
PubMed1994

Angiotensin II type 2 receptor stimulation of neuronal K+ currents involves an inhibitory GTP binding protein.

Angiotensin II (ANG II) elicits an ANG II type 2 (AT2) receptor-mediated increase in outward K+ current (IK; delayed rectifier K+ current) in neurons cocultured from rat hypothalamus and brain stem. Here we have shown that the AT2-receptor-mediated stimulation of neuronal IK by ANG II (100 nM) was abolished by pretreatment of cultures with pertussis toxin (PTX; 200 ng/ml) and by intracellular application of an antibody against the inhibitory guanine nucleotide (GTP) binding protein (anti-Gi alpha, 1:200). Antibodies against other GTP binding proteins (anti-Go alpha, 1:50 and 1:200; anti-Gq/11 alpha, 1:200) did not alter the AT2-receptor-mediated stimulation of neuronal IK by ANG II (100 nM). Furthermore, this effect of ANG II (100 nM) was inhibited by the serine/threonine phosphatase inhibitor okadaic acid (1-10 nM) and by anti-type 2A protein phosphatase (PP2A) antibodies but not by the tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). Thus we have identified key components (Gi and PP2A) of the signal transduction pathway that is responsible for the AT2-receptor-mediated stimulation of neuronal K+ currents.

Animals↗
PubMed1994

Calcium-modulated inward rectification of a calcium-activated potassium channel in neurons.

1. Inward rectification of a calcium-activated K+ channel in neurons cultured from the hypothalamus and brain stem of 1-day-old rats was studied by using patch-clamp techniques. A big conductance calcium-activated K+ channel with a slow gating rate was observed in inside-out patches. With symmetrical K+ across patches, inward conductance of this calcium-activated K+ channel was 216 +/- 14 (SE) pS (n = 4 patches), which changed little as different [Ca2+] was included in the bath solution. Outward conductance of this calcium-activated K+ channel was regulated by [Ca2+] in the bath solution and was 74 +/- 15 pS with 500 microM Ca2+. The higher level of Ca2+ on the intracellular side of the membrane caused the larger degree of rectification. Mg2+ only had a minor effect on rectification of this calcium-activated K+ channel.

Animals↗
PubMed1994

Magnesium and zinc potentiate ethanol inhibition of N-methyl-D-aspartate-stimulated nitric oxide synthase in cortical neurons.

The coupling of calcium mobilizing receptors to nitric oxide (NO) formation was examined in cerebral cortical cultures. Of the various agents tested, only glutamate, depolarization with KCl and the calcium ionophore ionomycin stimulated nitric oxide synthase (NOS) activity. Characterization of the glutamate response revealed that the ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA), kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic all stimulated NOS activity with a relative maximal efficacy of NMDA > kainate > alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic. Ethanol, Mg++ and Zn++ produced a concentration-dependent inhibition of NMDA stimulation of NOS. The Mg++ inhibition was reversed by increasing concentrations of NMDA, whereas Zn++ inhibition was not. Ethanol (100 mM) produced an apparent competitive type inhibition as seen by a parallel right-shift in the NMDA concentration-response curve. However, ethanol inhibition was dependent upon the presence of Mg++ and/or Zn++ in a concentration-related manner. Whereas 100 mM ethanol did not significantly inhibit NMDA stimulation of NOS activity in the absence of Mg++ and Zn++, inclusion of a combination of these cations increased the sensitivity to ethanol such that the NMDA response was completely blocked by 100 mM ethanol (IC50 approximately 30 mM). The potency for inhibition of NMDA stimulation of NOS by several short-chain alcohols followed their hydrophobicity profile and showed a similar dependency upon Mg++ for inhibition, alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic, but not kainate, stimulation of NOS was also inhibited by ethanol (100 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗