calcimycin and lucifer-yellow

We present electrical, physiological and molecular evidence for substantial electrical coupling of epithelial cells in Malpighian tubules via gap junctions. Current was injected into one principal cell of the isolated Malpighian tubule and membrane voltage deflections were measured in that cell and in two neighboring principal cells. By short-circuiting the transepithelial voltage with the diuretic peptide leucokinin-VIII we largely eliminated electrical coupling of principal cells through the tubule lumen, thereby allowing coupling through gap junctions to be analyzed. The analysis of an equivalent electrical circuit of the tubule yielded an average gap-junction resistance (R(gj)) of 431 kOmega between two cells. This resistance would stem from 6190 open gap-junctional channels, assuming the high single gap-junction conductance of 375 pS found in vertebrate tissues. The addition of the calcium ionophore A23187 (2 micromol l(-1)) to the peritubular Ringer bath containing 1.7 mmol l(-1) Ca(2+) did not affect the gap-junction resistance, but metabolic inhibition of the tubule with dinitrophenol (0.5 mmol l(-1)) increased the gap-junction resistance 66-fold, suggesting the regulation of gap junctions by ATP. Lucifer Yellow injected into a principal cell did not appear in neighboring principal cells. Thus, gap junctions allow the passage of current but not Lucifer Yellow. Using RT-PCR we found evidence for the expression of innexins 1, 2, 3 and 7 (named after their homologues in Drosophila) in Malpighian tubules. The physiological demonstration of gap junctions and the molecular evidence for innexin in Malpighian tubules of Aedes aegypti call for the double cable model of the tubule, which will improve the measurement and the interpretation of electrophysiological data collected from Malpighian tubules.

Topics: Aedes; Animals; Calcimycin; Dinitrophenols; Electric Impedance; Electrophysiology; Epithelial Cells; Gap Junctions; Gene Expression Regulation; Insect Proteins; Isoquinolines; Malpighian Tubules; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid

The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca(2+) ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53-66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Animals, Newborn; Calcimycin; Calcium; Calcium Signaling; Cells, Cultured; Central Nervous System; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Fluorescent Dyes; Gap Junctions; GAP-43 Protein; Gliosis; Ionophores; Isoquinolines; Microglia; Protein Kinase C; Rats; RNA, Messenger; Second Messenger Systems; Tetradecanoylphorbol Acetate; Up-Regulation

A new vesicle system is described that combines complementary properties of liposomes and polymeric beads. 'Lipobeads' consist of a lipid bilayer shell anchored on the surface of a hydrogel polymer cores which acts like a cytoskeleton. Anchoring is provided by fatty acids covalently attached to the surface of the hydrogel. These hydrophobic chains drive spontaneous assembly of a lipid bilayer shell around the modified hydrogel bead when exposed to a suspension of liposomes. The bilayer is stable and acts as a permeability barrier to compound loaded by prior absorption into the polymer core. Lipid mobility in the shell is similar to that found in other unanchored lipid bilayers. The system has potential application in drug delivery and for functional reconstitution of membrane proteins.

Topics: Acylation; Calcimycin; Calcium; Diffusion; Fluorescence; Isoquinolines; Lipid Bilayers; Liposomes; Microscopy, Confocal; Microspheres; Permeability; Polyvinyl Alcohol

To investigate in bovine and embryonic chicken lens cultures the effects of elevated intracellular calcium on the permeability of gap junctions. To determine the changes in intracellular calcium using fura-2. To detect any changes in the phosphorylation of connexin43 after ionophore treatment.. Lucifer yellow was micro-injected into individual cells, and dye spread to neighboring cells was evaluated. Intracellular calcium levels were measured using the calcium indicator, fura-2. Cultures were also labeled with 32P-orthophosphate followed by immunoprecipitation with antibodies specific for the gap junction protein, connexin43.. Bovine lens cultures incubated in the presence of either A23187 or ionomycin showed a reduction in intercellular dye transfer. The intracellular calcium concentrations in bovine cells were increased from a mean value of 0.11 +/- .009 microM in the controls to a mean of 0.40 +/- .073 microM with ionomycin treatment. Subsequent addition of EGTA to the medium decreased the intracellular calcium concentrations to a mean of 0.26 +/- .113 microM and reversed the inhibition of dye spread found with ionomycin. With ionomycin in the medium, the phosphorylated form of connexin43 was not as prominent as in the controls. In contrast, these same treatments had no detectable effect on junctional permeability in the embryonic chicken lens cultures. Dye spread was equally extensive and rapid under control and ionophore conditions, even though fura studies showed an elevation in intracellular calcium levels.. In the bovine cultures, physiologically relevant changes in the levels of cytoplasmic calcium markedly reduced dye transfer. The increase in cytoplasmic calcium was correlated with a change in the phosphorylation level of connexin43. The regulation of junctional communication in the chick lens cultures appears to differ significantly from that in the bovine system.

Topics: Animals; Calcimycin; Calcium; Cattle; Cell Communication; Cell Membrane Permeability; Cells, Cultured; Chick Embryo; Connexin 43; Fura-2; Gap Junctions; Ionomycin; Isoquinolines; Lens, Crystalline; Phosphorylation

The effect of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein, teleocidin, anthralin, the Ca2+-ionophore A23187, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT) and phenobarbital (PB) on lucifer yellow transfer in cultures of SV-40-transformed Djungarian hamster fibroblasts was studied. TPA, mezerein, teleocidin, A23187, DDT and BHT exerted a strong inhibitory effect on cell-to-cell dye transfer. Anthralin uncoupled cells in 3 experiments out of 6. PB appeared to enhance lucifer yellow transfer. Sodium nitrite, a substance with unknown promoting activity, effectively uncoupled cells. All the promoters investigated had a reversible effect on the dye transfer. The value of the dye transfer method for promoter screening is discussed.

Topics: Animals; Anthralin; Butylated Hydroxytoluene; Calcimycin; Carcinogens; Cells, Cultured; Cricetinae; DDT; Diterpenes; Fluorescent Dyes; Intercellular Junctions; Isoquinolines; Lyngbya Toxins; Phenobarbital; Terpenes; Tetradecanoylphorbol Acetate

The effects of tumor promoter--12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein, anthralin, Ca2+-ionophore A23187, butylated hydroxytoluene (BHT), DDT and phenobarbital--on cell-to-cell exchange of Lucifer Yellow were studied in cultures of SV40-transformed Djungarian hamster fibroblasts. TPA, mezerein, A23187, DDT and BHT strongly inhibited cell-to-cell exchange of Lucifer Yellow. Anthralin uncoupled cells in 3 out of 6 experiments. Phenobarbital, in contrast to other promoters, enhanced dye transfer. The effects of all the promoters tested were fully reversible. The potential use of Lucifer Yellow exchange inhibition as a test for the screening of tumor promoters is discussed.

Topics: Animals; Anthralin; Butylated Hydroxytoluene; Calcimycin; Carcinogens; Cell Line; Cricetinae; DDT; Diterpenes; Fluorescent Dyes; In Vitro Techniques; Isoquinolines; Phenobarbital; Terpenes; Tetradecanoylphorbol Acetate

Topics: Animals; Calcimycin; Calcium; Cell Line, Transformed; Cricetinae; Fibroblasts; Fluorescent Dyes; Intercellular Junctions; Isoquinolines; Protein Kinase C; Tetradecanoylphorbol Acetate

Single islet cells in monolayer cultures of neonatal rat pancreas were microinjected with the fluorescent dye Lucifer Yellow CH and the cultures were observed by combined phase contrast and fluorescent microscopy. The dye spread from an injected cell directly into neighboring islet cells, and successive microinjections of dye into different cells defined territories comprised of 2-6 communicating cells. The number of communicating cells could be modulated by addition to the cultures of different agents known to affect islet cell secretory activity. Cell communication was significantly increased by a high (16.7 mM) glucose concentration, by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM), and by the calcium ionophore, A23187. The effect of A23187 was transient and dose-dependent. Somatostatin (1 microgram/ml) significantly inhibited cell communication. These results demonstrate that cell-to-cell communication may participate in the regulation of islet cell secretory activity.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcimycin; Cell Communication; Cells, Cultured; Glucose; Islets of Langerhans; Isoquinolines; Rats; Somatostatin

The cells of Necturus gallbladder epithelium are electrically coupled. This work used intracellular injection of the fluorescent dye Lucifer yellow to demonstrate that these cells are also dye coupled and that this coupling is rapidly and reversibly inhibited by high concentrations of carbon dioxide. Dye coupling is also inhibited by the calcium ionophore A23187.

Topics: Animals; Calcimycin; Carbon Dioxide; Electric Conductivity; Fluorescent Dyes; Gallbladder; In Vitro Techniques; Isoquinolines; Necturus