Biomedical subjects

Roger J Thompson

Publications and source records attributed to Roger J Thompson.

5 recordsLinked to original sources

PubMed2006

Ischemia opens neuronal gap junction hemichannels.

Neuronal excitotoxicity during stroke is caused by activation of unidentified large-conductance channels, leading to swelling and calcium dysregulation. We show that ischemic-like conditions [O(2)/glucose deprivation (OGD)] open hemichannels, or half gap junctions, in neurons. Hemichannel opening was indicated by a large linear current and flux across the membrane of small fluorescent molecules. Single-channel openings of hemichannels (530 picosiemens) were observed in OGD. Both the current and dye flux were blocked by inhibitors of hemichannels. Therefore, hemichannel opening contributes to the profound ionic dysregulation during stroke and may be a ubiquitous component of ischemic neuronal death.

Adenosine Triphosphate↗

PubMed2006

Anion channels transport ATP into the Golgi lumen.

Anion channels provide a pathway for Cl(-) influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H(+)-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K(2)ATP added to the cis-chamber directly inhibited the Cl(-) subconductance levels of both channels, with K(m) values ranging from 16 to 115 microM. Substitution of either K(2)ATP or MgATP for Cl(-) on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl(-) > ATP(4-) > MgATP(2-). Single-channel currents were observed at physiological concentrations of Cl(-) and ATP, providing evidence for their importance in vivo. Second, transport of [alpha-(32)P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent K(m) = 25 microM. Finally, after transport of [gamma-(32)P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [alpha-(32)P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl(-) and ATP influx into the Golgi lumen.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

O2-sensitive K+ channels in immortalised rat chromaffin-cell-derived MAH cells.

The regulation of K(+) channels by O(2) levels is a key link between hypoxia and neurotransmitter release in neuroendocrine cells. Here, we examined the effects of hypoxia on K(+) channels in the immortalised v-myc, adrenal-derived HNK1(+) (MAH) cell line. MAH cells possess a K(+) conductance that is sensitive to Cd(2+), iberiotoxin and apamin, and which is inhibited by ~24 % when exposed to a hypoxic perfusate (O(2) tension 20 mmHg). This conductance was attributed to high-conductance Ca(2+)-activated K(+) (BK) and small-conductance Ca(2+)-activated K(+) (SK) channels, which are major contributors to the O(2)-sensitive K(+) conductance in adrenomedullary chromaffin cells. Under low [Ca(2+)](i) conditions that prevented activation of Ca(2+)-dependent K(+) conductances, a rapidly activating and slowly inactivating K(+) conductance, sensitive to both TEA and 4-aminopyridine (4-AP), but insensitive to 100 nM charybdotoxin (CTX), was identified. This current was also reduced (by ~25 %) when exposed to hypoxia. The hypoxia-sensitive component of this current was greatly attenuated by 10 mM 4-AP, but was only slightly reduced by 10 mM TEA. This suggests the presence of delayed-rectifier O(2)-sensitive channels comprising homomultimeric Kv1.5 or heteromultimeric Kv1.5/Kv1.2 channel subunits. The presence of both Kv1.5 and Kv1.2 alpha-subunits was confirmed using immunocytochemical techniques. We also demonstrated that these K(+) channel subunits are present in neonatal rat adrenomedullary chromaffin cells in situ. These data indicate that MAH cells possess O(2)-sensitive K(+) channels with characteristics similar to those observed previously in isolated chromaffin cells, and therefore provide an excellent model for examining the cellular mechanisms of O(2) sensing in adrenomedullary chromaffin cells.

Adrenal Glands↗

PubMed2002

Developmental regulation of O(2) sensing in neonatal adrenal chromaffin cells from wild-type and NADPH-oxidase-deficient mice.

In mammals, adrenomedullary chromaffin cells (AMC) express a neutrophil-like NADPH oxidase and secrete catecholamines, which play a vital role in the ability of the neonate to survive hypoxic stress. To test whether NADPH oxidase functions as an O(2) sensor, and whether mouse AMC express a developmentally regulated O(2)-sensing mechanism similar to rats, we compared the effects of hypoxia on cultured AMC from wild-type (WT) and transgenic oxidase-deficient (OD) mice, lacking the gp91(phox) subunit of NADPH oxidase. Hypoxia ( pO(2) congruent with5 mmHg) caused a reversible inhibition of outward K(+) current by congruent with27% ( n=6) in WT and congruent with29% ( n=9) in OD neonatal (P1-P5) chromaffin cells. O(2)-sensitive K(+) currents included both Ca(2+)-dependent ( I(BK)) and a delayed rectifier-like K(+) current ( I(KV)). Additionally, hypoxia depolarized WT and OD chromaffin cells and caused reversible broadening of the action potential. Exposure of both WT and OD neonatal AMC cultures to hypoxia (5% O(2)) for congruent with1 h caused four- to sixfold stimulation of catecholamine (CA) secretion as determined by HPLC. In contrast, hypoxia had no significant effect on K(+) currents or CA secretion in juvenile (P14-P15) AMC. Thus, mouse AMC possess a developmentally regulated O(2)-sensing mechanism, but NADPH oxidase does not function as the primary O(2)sensor in these cells.

Action Potentials↗

PubMed2002

A large-conductance anion channel of the Golgi complex.

An acidic lumenal pH is vital for the proper posttranslational modifications and sorting of proteins and lipids from the Golgi complex. We characterized ion channels present in Golgi fractions that have been cleared of transiting proteins. A large conductance anion channel was observed in approximately 30% of successful channel incorporations into the planar lipid bilayer. The channel, GOLAC-2, has six levels (one closed and five open). The open states are each approximately 20% increments of the maximal, 325 pS conductance. The channel was approximately 6 times more selective for Cl(-) over K(+). Binomial analysis of percent occupancy for each conducting level supports the hypothesis of five independent conducting pathways. The conducting levels can coordinately gate because full openings and closings were often observed. Addition of 3 to 5 mM reduced glutathione to the cis chamber caused dose-dependent increases in single channel conductance, indicating that the channel may be regulated by the oxidation-reduction state of the cell. We propose that GOLAC-2 is a co-channel complex consisting of five identical pores that have a coordinated gating mechanism. GOALC-2 may function as a source of counter anions for the H(+)-ATPase and may be involved in regulating charge balance and membrane potential of the Golgi complex.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗