No abstract

Gap junctions and the intercellular communication syncytium they form between glial cells are thought to play a critical role in glial maintenance of appropriate metabolic environments in neural tissues. We have previously suggested (Yamamoto et al., Brain Res. 508:313-319, '90) that the vast majority of astrocytes in rat brain express connexin43, one of several recently identified gap junction proteins. Here, we confirm ultrastructurally that astrocytes in a number of brain regions of rat are immunolabelled with an antibody against connexin43 and that neurons and oligodendrocytes are devoid of labelling. The distribution of connexin43 immunoreactivity throughout the brain is presented at the light microscope (LM) level. By LM, immunoreactive structures consisted primarily of round or elongated puncta ranging from 0.3 microns to 4 microns in length and of annular profiles ranging from 1 to 10 microns in diameter. Immunolabelled fibrous processes were only occasionally seen and no labelling was observed in astrocytic cell bodies. Long, linear arrays of puncta were rare in gray matter but were common in white matter where they were arranged parallel to myelinated fibers. Puncta organized in a honeycomb pattern were seen near the cerebral cortical surface and frequently around blood vessels. Regional immunoreaction density, which was a reflection of either the concentration or staining intensity of immunoreactive elements, was remarkably heterogeneous; dramatic differences in labelling intensity frequently delineated anatomical boundaries between adjacent nuclei. Abrupt as well as graded fluctuations of immunoreaction intensity were also observed within nuclear structures. By electron microscopy (EM), gap junctions of fibrous and protoplasmic astrocytes were intensely stained and labelled organelles were often observed intracellularly in areas near gap junctions. These junctions and the spread of immunoreaction product to perijunctional organelles in their vicinity were considered to correspond to puncta seen by LM. Labelling within astrocytic cell bodies was seen in only a few instances. In some brain areas, astrocytic processes commonly gave rise to immunoreactive lamellae that partially ensheathed neuronal cell bodies, axon terminals, dendrites, and synaptic glomeruli. Such lamellae were considered to correspond to immunoreactive annular profiles seen by LM. Perivascular endfoot processes of astrocytes displayed intense staining of their gap junctions and portions of their apposing membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane-permeant cAMP derivatives (dibutyryl-and 8-bromo-cAMP) increase gap-junctional conductance within minutes when applied to voltage-clamped pairs of rat hepatocytes. Glucagon also increases junctional conductances, but the response has a more rapid onset and is more rapidly reversible. The glucagon effect can be prevented by intracellular injection of the protein inhibitor of the cAMPdependent protein kinase (Walsh inhibitor), indicating that the catalytic subunit of cAMP-dependent protein kinase is directly involved. The 27-kDa miijor gap junction polypeptide is phosphorylated when liver cells dissociated into small groups are incubated with 32p. Addition of 8-bromo-cAMP to cells increases the incorporation of 32P into the 27-kDa junctional protein. Serine is the amino acid residue that is phosphorylated. When isolated liver gap junctions are incubated in the presence of catalytic subunit of the cAMP-dependent protein kinase, the 27-kDa gap junction polypeptide is phosphorylated with low stoichiometry on serine. The rapid increases in gap junctional conductance caused by agents that elevate cAMP and phosphorylation of the gap junction protein by cAMPdependent protein kinase suggest that cAMP-dependent phosphorylation of the gap junction channel modulates the conductance of liver gap junctions.

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Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junction and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by CAMP-dependent protein kinasc (CAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase I1 (Ca2+/CaM-PK 11) Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 3; demonstrated that CAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1 PKC also phosphorylated seryl residues in additional peptides. Ca2+/CaM-PK I1 phosphorylated serine and tc a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228 -239 based on the deduced amino acid sequence of rat connexiI 32) was phosphorylated by CAMP-PK and by PKC, with kinetic properties being similar to those for othei physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK I1 did not phosphorylate the peptide Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated thai Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by CAMP-PK or by PKC. In hepatocyte: labeled with [32P]~rthopho~phoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyratc (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the CAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.Gap junctions provide a pathway for cytoplasmic communication between adjacent cells [I, 21. These channels are permeable to ions and small molecules such as second messengers [3, 41. Hepatocyte gap junctions can be isolated by subcellular fractionation following detergent or alkaline extraction [5 -91. These preparations are highly enriched in proteins that have mobilities in the range of 26-28 kDa as determined by SDSjPAGE [7 -1 I]. Rat and human liver cDNAs have been isolated [lo, 111 which encode proteins having molecular masses of approximately 32 kDa. The rat or human liver proteins have been termed connexin 32 [12]. Rat liver also expresses a homologous protein of apparent molecular mass 21 kDa for which a cDNA has also been sequenced. Its predicted molecular mass is 26 kDa and it has been termed connexin ...