Extent of intercellular calcium wave propagation is related to gap junction permeability and level of connexin-43 expression in astrocytes in primary cultures from four brain regions

• Published in: Neuroscience Vol. 92; no. 1; pp. 255 - 265
• Main Authors: Blomstrand, F, Åberg, N.D, Eriksson, P.S, Hansson, E, Rönnbäck, L
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.01.1999; Elsevier
• Subjects: Animals; Astrocytes; Astrocytes - drug effects; Astrocytes - metabolism; Biological and medical sciences; Brain; Brain - cytology; Brain - drug effects; Brain - metabolism; Calcium; Calcium - metabolism; calcium waves; Cells; Cells, Cultured; Coloring Agents; Coloring Agents - pharmacokinetics; Connexin 43; Connexin 43 - genetics; Connexin 43 - metabolism; Cultured; cytology; drug effects; Extracellular Space; Extracellular Space - metabolism; Fundamental and applied biological sciences. Psychology; Fysiologi; Gap Junctions; Gap Junctions - metabolism; genetics; Glutamic Acid; Glutamic Acid - pharmacology; Immunohistochemistry; intercellular signaling; Isolated neuron and nerve. Neuroglia; MEDICAL AND HEALTH SCIENCES; MEDICIN OCH HÄLSOVETENSKAP; Messenger; metabolism; Neurologi; Neurology; Permeability; pharmacokinetics; pharmacology; Physiology; Rats; Rats, Sprague-Dawley; regional heterogeneity; RNA; RNA, Messenger - metabolism; Serotonin; Serotonin - pharmacology; Sprague-Dawley; Vertebrates: nervous system and sense organs; CI, confidence interval; EDTA, ethylenediaminetetra-acetate; SDS, sodium dodecyl sulfate; TEMED, N, N, N′, N′-tetramethylethylenediamine; MAP-2, microtubule-associated protein 2; PVP40-TTBS, polyvinylpyrrolidone-40-TTBS; 5-HT, 5-hydroxytryptamine; TTBS, Tween-20-Tris-buffered saline; regional heterogeneity; MEM, minimal essential medium; gap junctions; Ca 2+, calcium; GFAP, glial fibrillary protein; Cx-43, connexin-43; RPA, RNase protection assay; connexin-43; GJ, gap junction; HEPES, N-[2-hydroxyethyl]piperazine- N-[2-ethanesulfonic acid]; intercellular signaling; SD, spreading depression; calcium waves; TBE, Tris–boric acid–EDTA; 18α-GA, 18α-glycyrrhetinic acid; HHBSS, HEPES-buffered Hank's balanced salt solution; astrocytes; Calcium; Rat; Neuroglia; Rodentia; Central nervous system; Intercellular transport; Cell junction; Astrocyte; Permeability; Gap junction; Connexin; Signal transduction; Vertebrata; Mammalia; Brain (vertebrata)
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(98)00738-6

Astrocytes are coupled via gap junctions, predominantly formed by connexin-43 proteins, into cellular networks. This coupling is important for the propagation of intercellular calcium waves and for the spatial buffering of K +. Using the scrape-loading/dye transfer technique, we studied gap junction permeability in rat astrocytes cultured from four different brain regions. The cultures were shown to display regional heterogeneity with the following ranking of the gap junction coupling strengths: hippocampus=hypothalamus>cerebral cortex=brain stem. Similar relative patterns were found in connexin-43 messenger RNA and protein levels using solution hybridization/RNase protection assay and western blots, respectively. The percentages of the propagation area of mechanically induced intercellular calcium waves for cortical, brain stem and hypothalamic astrocytes compared with hippocampal astrocytes were approximately 77, 42, and 52, respectively. Thus, the extent of calcium wave propagation was due to more than just gap junctional permeability as highly coupled hypothalamic astrocytes displayed relatively small calcium wave propagation areas. Incubation with 5-hydroxytryptamine decreased and incubation with glutamate increased the calcium wave propagation area in hippocampal (67% and 170% of the control, respectively) and in cortical astrocytes (82% and 163% of the control, respectively). Contrary to hippocampal and cortical astrocytes, the calcium wave propagation in brain stem astrocytes was increased by 5-hydroxytryptamine incubation (158% of control), while in hypothalamic astrocytes, no significant effects were seen. Similar effects from 5-hydroxytryptamine or glutamate treatments were observed on dye transfer, indicating an effect on the junctional coupling strength. These results demonstrate a strong relationship between connexin-43 messenger RNA levels, protein expression, and gap junction permeability among astroglial cells. Furthermore, our results suggest heterogeneity among astroglial cells from different brain regions in intercellular calcium signaling and in its differential modulation by neurotransmitters, probably reflecting functional requirements in various brain regions.