Biomedical subjects

Colin Sumners

Publications and source records attributed to Colin Sumners.

9 recordsLinked to original sources

PubMed2003

PI3-kinase inhibitors abolish the enhanced chronotropic effects of angiotensin II in spontaneously hypertensive rat brain neurons.

Angiotensin II (Ang II), acting at Ang II type 1 receptors (AT1Rs), increases the firing rate of neurons from Wistar-Kyoto (WKY) rat brain via protein kinase C (PKC)- and calcium-calmodulin kinase II (CaMKII)-dependent mechanisms. The objectives of this study were twofold; first, to compare the Ang-II-stimulated increase in firing of neurons from WKY and spontaneous hypertensive rats (SHR) and second, to elucidate the signaling mechanisms involved. Action potentials were measured in neurons cultured from SHR and WKY rat brains using the whole cell configuration of the patch-clamp technique in the current-clamp mode. Ang II (100 nM) caused three- and sixfold increases in neuronal firing rate in WKY rat and SHR neurons, respectively; effects that were abolished by the AT1R antagonist Losartan (1 microM). Co-administration of calphostin C (10 microM, a PKC inhibitor) and KN-93 (10 microM, a CaMKII inhibitor) completely blocked this Ang II action in WKY rat neurons, while they caused only a approximately 50% attenuation in SHR neurons. The residual increase in firing rate produced by Ang II in SHR neurons was blocked by inhibitors of phosphatidylinositol 3 kinase (PI3-kinase), either LY 294002 (10 microM) or wortmannin (100 nM). These observations suggest that a PI3-kinase signaling pathway may be responsible for the enhanced chronotropic effect produced by Ang II in SHR neurons.

Action Potentials↗

PubMed2003

Drinking behavior elicited by central injection of angiotensin II: roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.

Angiotensin II↗

PubMed2003

Cytokine-stimulated inducible nitric oxide synthase expression in astroglia: role of Erk mitogen-activated protein kinase and NF-kappaB.

Expression of inducible nitric oxide synthase (iNOS), which leads to the production of nitric oxide (NO), is stimulated by proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Here we report on the roles of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases in IL-1beta/TNF-alpha-induced iNOS expression in adult rat astroglia. Cytokine-induced increases in nitrite accumulation (an index of NO production) and iNOS expression were attenuated by inhibition of NF-kappaB with pyrrolidine dithiocarbamate (PDTC). Similar attenuation of these cytokine-induced responses was produced by inhibition of MAP kinase (MEK), the immediate upstream activator of Erk, using PD098,059. Combined treatment of astroglia with PDTC and PD098,059 completely abolished the cytokine-induced increases in iNOS expression and nitrite accumulation. By contrast, the selective p38 kinase inhibitor SB203,580 amplified the effects of IL-1beta/TNF-alpha on nitrite accumulation. In accordance with these findings, IL-1beta- and TNF-alpha-induced a time-dependent increase in Erk1/Erk2 activation. This cytokine action was completely abolished by PD098,059 but was not altered by PDTC. Finally, IL-1beta and TNF-alpha induced degradation of NF-kappaB's bound inhibitory protein, IkappaB-alpha, leading to translocation of NF-kappaB into the nucleus. IkappaB-alpha expression was not restored to control levels by inhibition of MEK. Furthermore, inhibition of MEK with PD098,059 did not alter IL-1beta- and TNF-alpha-induced expression of active NF-kappaB. The results demonstrate that autonomous Erk and NF-kappaB pathways mediate cytokine-induced increases in iNOS expression in astroglia.

Animals↗

PubMed2002

Transduction of a functional domain of the AT1 receptor in neurons by HIV-Tat PTD.

Despite advances in transgenic and gene transfer technologies, in vivo structure-function studies of the angiotensin II type I receptor (AT1R) have revealed limited information on the diverse actions of angiotensin II. Our objective in the present study was to determine if protein transduction technology with the use of the HIV-Tat protein transduction domain could fill this gap. Recombinant HIV-Tat protein transduction domain fused to EGFP and to the third intracellular loop of the AT1R was expressed. Incubation of hypothalamus and brainstem neurons with this peptide indicated an efficient transport of the protein to most of the cells. This transduction was accompanied by an increase in neuronal firing rate, an effect similar to that observed with angiotensin II stimulation of the neuronal AT1R. The characteristics of the chronotropic effects of recombinant third intracellular loop and its synthetic counterpart were similar and comparable to the effects of angiotensin II on these neurons. In addition, in the presence of the protein kinase C inhibitor calphostin C, the peptide failed to increase firing rate. These observations demonstrated that transduction of neurons with the third intracellular loop of the AT1R produces chronotropic effects similar to those induced by angiotensin II. The data suggests that protein transduction technology could be useful for in vivo AT1R domain transduction.

Action Potentials↗

PubMed2002

Hypertension-linked decrease in the expression of brain gamma-adducin.

Gene profiling data coupled with adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of adducins in neurotransmission at the cellular level. All three adducin subunits (alpha, beta, and gamma) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the gamma-adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in gamma-adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize gamma-adducin gene expression. A hypertension-linked decrease of gamma-adducin was confirmed by demonstrating a decrease in gamma-adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a gamma-adducin-specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous gamma-adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in gamma-adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters.

Action Potentials↗

PubMed2002

Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem.

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker alpha-internexin, while the other stained with the astrocyte marker GFAP. The alpha-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These alpha-internexin positive cells coimmunostained for the neuronal markers MAP2, type III beta-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these alpha-internexin positive cells revealed small Ca(2+) currents with a peak current of -0.5 +/- 0.1 pA/pF at a membrane potential of -20 mV (n = 5) and half-maximal activation at -30 mV (n = 5). Na(+) currents with a peak current density of -154.5 +/- 49.8 pA/pF at a membrane potential of -15 mV (n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.

Age Factors↗

PubMed2002

Obligatory role of protein kinase Cbeta and MARCKS in vesicular trafficking in living neurons.

Neurotransmitter release from neurons involves both vesicular trafficking and subsequent fusion of synaptic vesicles with the plasma membrane. The mechanisms involving the formation and fusion of vesicles that allow the exocytotic release of transmitters are understood well. Little is known, however, about the signaling mechanism involved in the trafficking of vesicles along the neurites. In this study, we used real-time confocal microscopy to search for evidence that vesicular trafficking in neurons requires the activation of protein kinase Cbeta (PKCbeta) and the myristoylated alanine-rich C kinase substrate (MARCKS) signaling pathway. Dopamine-beta-hydroxylase fused to green fluorescent protein has been used to trace vesicular movement. Angiotensin II, an established neuromodulatory hormone, stimulates translocation of green fluorescent protein-dopamine-beta-hydroxylase vesicles from the cell body to neurites. This translocation was blocked by an antisense oligonucleotide to PKCbeta and MARCKS. Stimulation of PKC by other means, such as phorbol-12-myristate-13-acetate or carbachol, also resulted in the redistribution of fluorescence in a manner similar to that observed for angiotensin II. These observations demonstrate that PKCbeta-MARCKS signaling may be a general mechanism for the stimulation of vesicular trafficking in brain neurons.

Angiotensin II↗

PubMed2002

Chronotropic action of angiotensin II in neurons via protein kinase C and CaMKII.

Angiotensin II (Ang II) plays an important role in the central control of blood pressure and baroreflexes. These effects are initiated by stimulation of Ang II type 1 (AT(1)) receptors on neurons within the hypothalamus and brain stem, and involve increasing the activity of noradrenergic, substance P, and glutamatergic pathways. The goal of this study is to investigate the intracellular signaling molecules, which are involved in mediating the Ang II-induced increases in neuronal activity. Using neurons in primary culture from newborn rat hypothalamus and brain stem, we have previously determined that Ang II elicits an AT(1) receptor-mediated inhibition of delayed rectifier K(+) current, a stimulation of Ca(2+) current, and a consequent increase in firing rate. In the present study we have demonstrated that this chronotropic action of Ang II in neuronal cultures involves activation of Ca(2+)-dependent signaling molecules. The Ang II-induced increase in firing rate was abolished by inhibition of phospholipase C with U73122 (10 micromol/L), and was attenuated by the protein kinase C inhibitor calphostin C (10 micromol/L) or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 (10 micromol/L). A combination of calphostin C and KN-93 completely inhibited this Ang II action. These results indicate that the AT(1) receptor-mediated increase in neuronal firing rate involves activation of both PKC and CaMKII, and suggest that these enzymes are potential targets for manipulating the central actions of Ang II.

Angiotensin II↗

PubMedDate not supplied

Angiotensin AT1 receptor signalling pathways in neurons.

1. The aim of the present article is to review the intracellular signal transduction pathways that are influenced by the peptide angiotensin (Ang) II, acting via its type 1 (AT1) receptor, in neurons. 2. The AT1 receptors couple to a wide variety of signalling pathways in peripheral tissues, such as kidney, heart and vascular smooth muscle. A similar diversity of signalling mechanisms exists for AT1 receptors in neurons. 3. We outline the known neuronal AT1 receptor signalling pathways as they relate to function. Pathways that couple activation of AT1 receptors to short-term changes in neuronal membrane ionic currents and firing rate will be reviewed. These are different from the pathways that elicit longer-term changes in enzyme activity and gene expression and, ultimately, increases in noradrenaline synthesis. 4. Novel AT1 receptor signalling pathways discovered through gene expression profiling and their potential functional significance have been discussed.

Angiotensin II↗