Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell–cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from −60 to +100 mV; threshold was correlated with extracellular K⁺, so that at 10 mM K⁺, channels began to open at −30 mV. Evaluation of cell–cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10–20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K⁺ provides a mechanism for enhancement of ATP release under pathological conditions.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

Growth-suppressive function of human connexin32 in a conditional immortalized mouse hepatocyte cell line

Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Cx26 and Cx32 that characterizes normal mouse hepatocytes, expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes, we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39 degrees C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39 degrees C, growth rates of both parental cells and transfectants were of the same level. When examined at 37 degrees C, growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However, no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37 degrees C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.     

Slow intercellular Ca(2+) signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca(2+) waves that propagated with mean velocities of approximately 14 micrometer/s, reaching approximately 80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca(2+) waves, although the velocity and number of cells communicated by the Ca(2+) signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca(2+) signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P(2)-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca(2+) signals in wild-type neonatal mouse cardiac myocytes. Activation of P(2)-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca(2+) signals. The importance of such ATP-mediated Ca(2+) signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.     

Connexins Induce and Maintain Tight Junctions in Epithelial Cells

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)–independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na⁺/K⁺-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC–independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and –independent manners in epithelial cells.     

Endotoxin unmasks the role of gap junctions in the liver

Gap junctions are thought to be necessary for proper tissue function. However, no clear hepatic phenotype has been described in patients lacking connexin 32 (Cx32), the principal gap junction in liver. To determine the physiological role of Cx32 in liver, we compared the response of wild type and Cx32-deficient mice to endotoxin, since this stress increases serum levels of hormones that bind to receptors that are asymmetrically distributed across the hepatic lobule. In hepatocyte couplets isolated from wild type mice, most hepatocytes could transfer microinjected dye to their neighbor even after treatment with endotoxin. Dye transfer was not observed in Cx32-deficient couplets. Treatment of hepatocyte couplets from wild type mice with vasopressin induced calcium (Ca(2+)) waves that crossed the couplets in a concentration-dependent fashion, but the delay in transmission was markedly prolonged at all concentrations in Cx32-deficient couplets. Expression of the vasopressin receptor and the inositol 1,4,5-trisphosphate receptor was not decreased by endotoxin or in Cx32-deficient couplets. Finally, endotoxin caused transient hypoglycemia and cholestasis in wild type animals, but hypoglycemia was slightly prolonged and cholestasis was much worse in Cx32-deficient mice treated with endotoxin. The hepatic response to endotoxin is markedly impaired in the absence of Cx32. Thus, an important role of gap junctions in the liver is to assure integrated and uniform tissue response in times of stress.     

Gene expression alterations in connexin null mice extend beyond the gap junction

Connexin43 (Cx43) is the principal gap junction protein between astrocytes in the neonatal brain and also interconnects neural precursor cells during CNS development. In an attempt to understand global effects of expression of the Cx43 gap junction gene on development and function of the nervous system, we have compared gene expression patterns in cultured astrocytes and brains from wildtype mice with those in which Cx43 is deleted as well as in spinal cords of experimental autoimmune encepahlomyelitis (EAE) mice. One surprising result obtained from high densitity mouse cDNA studies was the large number of genes that were statistically altered in mice with decreased expression of Cx43. These altered genes encode proteins with a wide range of functions within cells, and thus deletion of normal gap junction expression appears to result in globally altered glial functions in addition to disruption of intercellular communication. Here we discuss those results in the context of the strategies and data analysis paradigms that we are using in such studies.     

Decreased gap-junctional communication associated with segregation of the neuronal phenotype in the RT4 cell-line family

RT4 is a family of cell lines derived from a rat peripheral neurotumor. The RT4 family consists of a multipotential stem-cell line that spontaneously gives rise to three derivative cell types, one glial and two neuronal. The three derivative cell types are capable of further lineage-specific maturation under appropriate culture conditions. Gap-junctional communication is postulated to be important during nervous-system development by allowing and/or controlling the transmission of both electrical current and signaling molecules, which may affect growth and differentiation. Our characterization of gap-junctional communication in the RT4 cell line family revealed that: (1) the glial-derivative and the stem-cell line were extensively coupled, while the two neuronal derivatives were significantly less coupled, and (2) all of the RT4 cell lines, including the stem-cell line, expressed Cx43 mRNA and protein, and the levels were generally consistent with the observed degree of functional coupling. These observations are consistent with data from in vivo studies and establish the RT4 cell line family as a potentially useful in vitro model system for understanding the role(s) of gap-junctional communication during differentiation in the peripheral nervous system.