Reduced gap junctional communication among astrocytes in experimental diabetes: contributions of altered connexin protein levels and oxidative-nitrosative modifications.

Experimental diabetes increases production of reactive oxygen-nitrogen species and inhibits astrocytic gap junctional communication in tissue culture and brain slices from streptozotocin (STZ)-diabetic rats by unidentified mechanisms. Relative connexin (Cx) protein levels were assessed by Western blotting using extracts from cultured astrocytes grown in high (25 mmol/liter) or low (5.5 mmol/liter) glucose for 2-3 weeks and STZ-diabetic rat brain. Chemiluminescent signals for diabetic samples were normalized to those of controls on the same blot and same protein load. Growth in high glucose did not alter relative Cx26 level, whereas Cx30 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were reduced by ∼30%, and Cx43 increased ∼1.9-fold. In the inferior colliculus of STZ-diabetic rats, Cx30 and Cx43 levels in three of four rats were half those of controls, whereas GAPDH and actin were unaffected. Diabetes did not affect levels of Cx30, Cx43, or GAPDH in cerebral cortex, but actin level rose 24%. Cx43 was predominantly phosphorylated in control and diabetic samples, so the reduced dye transfer is not due to overall dephosphorylation of Cx43. Astrocytic growth in high glucose reduced the dye-labeled area by 75%, but 10 min of treatment with dithiothreitol restored normal dye transfer. In contrast, nitric oxide donors inhibited dye transfer among astrocytes grown in low glucose by 50-65% within 1 hr. Thus, modifications arising from oxidative-nitrosative stress, not altered connexin levels, may underlie the reduced dye transfer among severely hyperglycemic cultured astrocytes, whereas both oxidative-nitrosative stress and regionally selective down-regulation of connexin protein content may affect gap junctional communication in the brains of STZ-diabetic rats.

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons.

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.

Selective astrocytic gap junctional trafficking of molecules involved in the glycolytic pathway: impact on cellular brain imaging.

To assess the specificity of metabolite trafficking among gap junction-coupled astrocytes, we developed novel, real-time, single-cell enzymatic fluorescence assays to assay cell-to-cell transfer of unlabeled glycolytic intermediates and report (i) highly restricted transfer of glucose-6-phosphate (P) and two analogs, deoxyglucose (DG)-6-P, and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG-6-P, compared with DG and 2- and 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG, (ii) extensive junctional diffusion of glyceraldehyde-3-P, NADH, and NADPH plus three anionic fluorescent dyes used as internal standards for transfer assays, and (iii) stimulation of gap junctional communication by increased intracellular Na(+) that also evokes metabolic responses in nearby coupled astrocytes. Thus, dye transfer does not predict gap junctional permeability of endogenous metabolites. Intracellular retention of flux-regulating compounds (e.g. glucose-6-P) may be necessary for local metabolic control, whereas 'syncytial sharing' may dissipate the work load on peri-synaptic astrocytes. Imaging of brain functional activity depends on local accumulation of exogenous or endogenous signals, and DG-6-P is trapped in the cell where it is phosphorylated, whereas rapid dispersal of cytoplasmic NAD(P)H and labeled glucose metabolites throughout the astrocytic syncytium can interfere with cellular assessment of neuron-astrocyte relationships in autoradiographic, fluorescence microscopic, and magnetic resonance spectroscopic studies.

Protein kinase Czeta regulates phospholipase D activity in rat-1 fibroblasts expressing the alpha1A adrenergic receptor.

BACKGROUND: Phenylephrine (PHE), an alpha1 adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing alpha1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCzeta to PLD activation in response to PHE in these cells. RESULTS: PHE stimulated a PLD activity as demonstrated by phosphatidylethanol production. PHE increased PKCzeta translocation to the particulate cell fraction in parallel with a time-dependent decrease in its activity. PKCzeta activity was reduced at 2 and 5 min and returned to a sub-basal level within 10-15 min. Ectopic expression of kinase-dead PKCzeta, but not constitutively active PKCzeta, potentiated PLD activation elicited by PHE. A cell-permeable pseudosubstrate inhibitor of PKCzeta reduced basal PKCzeta activity and abolished PHE-induced PLD activation. CONCLUSION: alpha1A adrenergic receptor stimulation promotes the activation of a PLD activity by a mechanism dependent on PKCzeta; Our data also suggest that catalytic activation of PKCzeta is not required for PLD stimulation.

Hyperglycaemia and diabetes impair gap junctional communication among astrocytes.

Sensory and cognitive impairments have been documented in diabetic humans and animals, but the pathophysiology of diabetes in the central nervous system is poorly understood. Because a high glucose level disrupts gap junctional communication in various cell types and astrocytes are extensively coupled by gap junctions to form large syncytia, the influence of experimental diabetes on gap junction channel-mediated dye transfer was assessed in astrocytes in tissue culture and in brain slices from diabetic rats. Astrocytes grown in 15-25 mmol/l glucose had a slow-onset, poorly reversible decrement in gap junctional communication compared with those grown in 5.5 mmol/l glucose. Astrocytes in brain slices from adult STZ (streptozotocin)-treated rats at 20-24 weeks after the onset of diabetes also exhibited reduced dye transfer. In cultured astrocytes grown in high glucose, increased oxidative stress preceded the decrement in dye transfer by several days, and gap junctional impairment was prevented, but not rescued, after its manifestation by compounds that can block or reduce oxidative stress. In sharp contrast with these findings, chaperone molecules known to facilitate protein folding could prevent and rescue gap junctional impairment, even in the presence of elevated glucose level and oxidative stress. Immunostaining of Cx (connexin) 43 and 30, but not Cx26, was altered by growth in high glucose. Disruption of astrocytic trafficking of metabolites and signalling molecules may alter interactions among astrocytes, neurons and endothelial cells and contribute to changes in brain function in diabetes. Involvement of the microvasculature may contribute to diabetic complications in the brain, the cardiovascular system and other organs.

An anatomic mathematical measurement to find an adequate recipient M4 branch for superficial temporal artery to middle cerebral artery bypass surgery.

OBJECTIVE: With the recent interest in superficial temporal artery-middle cerebral artery (MCA) bypass for hemodynamic related ischemia, we performed an anatomic study to find the best possible craniotomy site that will allow finding a suitable recipient cortical artery without compromising the use of the best branch and/or segment of the donor's superficial temporal artery branches. METHODS: Anatomic dissection and measurements were performed in 15 injected cadaveric heads to verify the location of an adequate temporal recipient cortical branch of the MCA. The location of the branch was then correlated with surface anatomic landmarks. Mathematical measurements were then derived. RESULTS: A perpendicular line measuring 5 cm in length is drawn starting from a point two-thirds the distance from the lateral canthus to the tragus, and ending at the center of a circle measuring 3 cm in diameter, which is equivalent to the craniotomy size and site. This craniotomy will expose the posterior aspect of the sylvian fissure and exposes no less than two M4 temporal MCA branches. The diameter of at least one branch is larger than 1 mm in 93% of the specimens. These findings were later successfully applied to several bypass operations. CONCLUSION: This study provides an anatomic and patient-independent mathematical measurement as a way to predictably find an adequate recipient temporal M4 branch for superficial temporal artery-MCA bypass in the majority of patients.

Astrocytic connexin distributions and rapid, extensive dye transfer via gap junctions in the inferior colliculus: implications for [(14)C]glucose metabolite trafficking.

The inferior colliculus has the highest rates of blood flow and metabolism in brain, and functional metabolic activity increases markedly in response to acoustic stimulation. However, brain imaging with [1- and 6-(14)C]glucose greatly underestimates focal metabolic activation that is readily detected with [(14)C]deoxyglucose, suggesting that labeled glucose metabolites are quickly dispersed and released from highly activated zones of the inferior colliculus. To evaluate the role of coupling of astrocytes via gap junctions in dispersal of molecules within the inferior colliculus, the present study assessed the distribution of connexin (Cx) proteins in the inferior colliculus and spreading of Lucifer yellow from single microinjected astrocytes in slices of adult rat brain. Immunoreactive Cx43, Cx30, and Cx26 were heterogeneously distributed; the patterns for Cx43 and Cx 30 differed and were similar to those of immunoreactive GFAP and S100beta, respectively. Most Cx43 was phosphorylated in resting and acoustically stimulated rats. Dye spreading revealed an extensive syncytial network that included thousands of cells and perivasculature endfeet; with 8% Lucifer yellow VS and a 5-min diffusion duration, about 6,100 astrocytes (range 2,068-11,939) were labeled as far as 1-1.5 mm from the injected cell. The relative concentration of Lucifer yellow fell by 50% within 0.3-0.8 mm from the injected cell with a 5-min diffusion interval. Perivascular dye labeling was readily detectable and often exceeded dye levels in nearby neuropil. Thus, astrocytes have the capability to distribute intracellular molecules quickly from activated regions throughout the large, heterogeneous syncytial volume of the inferior colliculus, and rapid trafficking of labeled metabolites would degrade resolution of focal metabolic activation.

Calcium and protein kinase C (PKC)-related kinase mediate alpha 1A-adrenergic receptor-stimulated activation of phospholipase D in rat-1 cells, independent of PKC.

A previous study conducted in rat-1 cells expressing alpha(1A)-adrenergic receptors showed that phenylephrine (PHE) stimulates phospholipase D (PLD) activity. This study was conducted to determine the contribution of protein kinase C (PKC) to PHE-induced PLD activation in these cells. PKC inhibitors bisindolylmaleimide (BIM) I and Ro 31-8220, but not Gö 6976 or a pseudosubstrate peptide inhibitor of PKCalpha, decreased PLD activity and arachidonic acid release elicited by PHE. However, antisense oligonucleotides directed against PKC alpha, delta, epsilon, and eta reduced PKC isoform levels by about 80% but failed to alter PHE-induced PLD activation, indicating that these PKC isoforms are not involved in PLD activation elicited by alpha1A-adrenergic receptor stimulation. Ectopic expression of a kinase-deficient mutant of the PKC-related kinase PKN significantly attenuated PHE-induced PLD activation. On the other hand, BIM I and Ro 31-8220 blocked PHE-mediated increase in intracellular Ca2+ but Gö 6976 and the peptide inhibitor did not. In the absence of extracellular Ca2+, PHE failed to increase PLD activity. These results indicate that alpha1A-adrenergic receptor-stimulated PLD activation is mediated by a mechanism independent of PKCalpha, delta, epsilon, and eta, but dependent on a PKC-related kinase, PKN. Moreover, PKC inhibitors BIM I and Ro 31-8220 block PHE-induced PLD activity by inhibiting calcium signal. Caution should be used in interpreting the data obtained with PKC inhibitors in vivo.