We have recently reported that liposomes in combination with histidine (HK)-containing polymers enhanced the expression of luciferase in transfected cells. In transformed or malignant cell lines, branched HK polymers (combined with liposome carriers) were significantly more effective than the linear HK polymer in stimulating gene expression. In the current study, we found that the linear HK polymer enhanced gene expression in primary cell lines more effectively than the branched polymers. The differences in the optimal carrier (linear versus branched) were not due to initial cellular uptake, size of the complexes or level of gene expression. There was, however, a strong association between the optimal type of HK polymer and the pH of endocytic vesicles (P = 0.0058). By altering the percentage of histidines carrying a positive charge, the endosomal pH of a cell may determine the amount of DNA released from the linear or branched HK polymer. In the two cell lines in which the linear HK was the optimal polymer, the endocytic vesicles were strongly acidic with a pH of <5.0. Conversely, in the four cell lines in which the branched polymers were optimal transfection agents, the pH of endocytic vesicles was >6.0. Furthermore, binding data support the relationship between DNA release from the optimal HK polymer and endosomal pH. The interplay between optimal HK polymers and the endosomal pH may lead to improved gene-delivery polymers tailored to a particular cell.

The development of motility in cultured cells is usually associated with a polarization of the cell shape. In particular, the leading edge of the cell is extended into a lamella which acts as a locus for the elaboration of cell processes and for the formation of cell-substrate contacts and, at the opposite end, retraction fibres often extend beyond the trailing edge of the cell. The alignment of microfilament bundles (stress fibres) along the direction of migration and the presence of a band of actin at the leading edge of the cell suggest an involvement of this protein in the motile process. The direction of growth and orientation of various cell types in tissue culture can be influenced by externally applied d.c. electric fields but the effect of the field on cellular motile activities is unknown. Here we describe a galvanotropic response of cultured Xenopus epithelial cells. At a field strength of 5 V cm-1 these cells elongate perpendicularly with respect to the field. The anodal side of the cell retracts and both the ends and cathodal edge become active in the extension of ruffling lamellipodia. In parallel with the change in the cell axis, stress fibres are oriented perpendicularly to the field, and a band of actin is associated with the lamellae at the cathodal edge and at the ends of the cell.

Confocal laser scanning microscopy was used to visualize intercellular transmission of Ca2+ waves in intact rat ventricular trabeculae micro‐injected with the calcium indicator fluo‐3. Ca2+ waves usually failed to be transmitted from cell to cell. At identified individual end‐to‐end cell contacts, successful transmission interspersed with failure, which sometimes occurred despite an apparent small spritz of Ca2+ between cells. The probability of cell to cell transmission (Ptran) was 0.13. Ca2+ waves arose preferentially near junctions of connected cells, where connexin‐43 was found, but randomly in enzymatically disconnected heart cells. β‐Adrenergic stimulation significantly increased Ptran (to 0.22) and heptanol, an uncoupler of gap junction channels, significantly decreased it (to 0.045). In regions of high [Ca2+]i due to damage, wave frequency decreased markedly with each cell‐cell junction passed. The Ca2+ permeability of cardiac gap junctions may be regulated, and the low ability of cardiac gap junctions to transmit Ca2+ may help control the spread of Ca2+ from damaged regions.

Regulation of GABA release is crucial for normal brain functioning, and GABAA-mediated IPSCs are strongly influenced by repetitive stimulation and neuromodulation. However, GABA exocytosis has not been examined directly in organized tissue. Important issues remain outside the realm of electrophysiological techniques or are complicated by postsynaptic factors. For example, it is not known whether all presynaptic modulators affect release from all boutons in the same way, or whether modulator effects depend on the presence of certain types of voltage-gated calcium channels (VGCCs). To address such issues, we used confocal imaging and styryl dyes to monitor exocytosis from identified GABAergic boutons in organotypic hippocampal slice cultures. Repetitively evoked IPSCs declined more rapidly and completely than exocytosis, suggesting that depletion of filled vesicles cannot fully account for IPSC depression and underscoring the usefulness of directly imaging exocytosis. Stimulation at 10 Hz produced a transient facilitation of exocytosis that was dependent on L-type VGCCs. Using specific toxins, we found that release mediated via N-type and P-type VGCCs had similar properties. Neither baclofen nor a cannabinoid receptor agonist, CP55940, affected all boutons uniformly; they slowed release from some but completely prevented detectable release from others. Increasing stimulus frequency overcame this blockade of release. However, baclofen and CP55940 did not act identically, because only baclofen reduced facilitation and affected bouton releasing via P/Q-type VGCCs. Direct observation thus revealed novel features of GABAergic exocytosis and its regulation that would have been difficult or impossible to detect electrophysiologically. These features advance the understanding of the regulation of synapses and networks by presynaptic inhibition.