The electroporation metiod has been shown to be usefuil technique for introducing DNA into mammalian cells (1). It is believed that cell poration is caused by reversible electrical breakdown of the cell membrane (2). Previously, we have shown that upon electroporation the cells are sensitive to the osmolarity (3) and ionic composition of the medium. A similar conclusion was reached for chemically permeabilized cells which can be successfully maintained in buffers that resemble the intracellular ionic composition (cytomix: 120 mM KCl; 0.15 mM CaCl2; 10 mM K2HPO4/KH2PO4, pH 7.6; 25 mM Hepes, pH 7.6; 2 mM EGTA, pH 7.6; 5mM MgCl2; pH adjusted with KOH), and used for metabolic studies (4). Shortly before use freshly prepared ATP (2 mM; pH = 7.6 adjusted with KOH) and glutathione (5 mM) were added to prevent leakage of the cytoplasmic components, protect membranes against oxidation and facilitate resealing of pores (5: A.J.Meijer, personal communication).
Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (Mr, 40000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Doublelabeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx4O-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx4O -expressing myocytes are localized in the conduction system, ie, the His bundle, the bundle branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx4O is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Doublelabeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx4O antibodies demonstrated that, in tissues expressing both Cx43 and Cx4O, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins. (Circ Res. 1994;74:839-851
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