carbocyanines and indo-1

The ability to simultaneously monitor different myenteric neurones in a multilayer preparation may enhance our understanding of the enteric nervous system. Longitudinal muscle myenteric plexus preparations were mounted in recording chambers with a coverslip base and loaded with Indo-1-AM. cytosolic Ca2+ concentration ([Ca2+]i); changes were recorded at room temperature with a confocal microscope. In addition to mechanical (pressure-ring) and pharmacological (nifedipine) reduction of muscle contractions, purpose-designed software was developed to reposition regions of interest and avoid artefacts. Confocal scanning permitted optical selection of single cell layers. High K+ depolarization, used to distinguish between excitable and nonexcitable cells, caused a synchronous [Ca2+]i rise in 84.3% of the ganglion cells. Acetylcholine, substance P and serotonin (all at 10(-5) mol L(-1)) induced transient [Ca2+]i changes in subpopulations of myenteric neurones (45.1%, 42.9 and 21.9%, respectively). In addition to immediate responses to agonists, delayed [Ca2+]i changes were also recorded, suggesting the presence of both directly activated and synaptically driven neurones. Functionally identified neurones and other cells in close apposition to the ganglia (interstitial cells of Cajal) could also be studied. This study demonstrates the potential of optical Ca2+ recordings to monitor spread of activity in myenteric neurones and to study their interaction with non-neuronal targets.

Topics: Acetylcholine; Animals; Calcium; Calcium Channel Blockers; Carbocyanines; Cytosol; Electrophysiology; Female; Fluorescent Dyes; Free Radical Scavengers; Guinea Pigs; Indoles; Intestines; Male; Microscopy, Confocal; Motor Neurons; Muscle, Smooth; Myenteric Plexus; Nifedipine; Potassium; Pressure; Serotonin; Substance P; Vasodilator Agents

A brief exposure to high concentrations of glutamate kills cultured forebrain neurons by an excitotoxic process that is dependent on Ca2+ influx through the NMDA receptor. In this study, we have measured striking changes in mitochondrial function during and immediately after intense glutamate receptor activation. Using indo-1 microfluorometry and a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP-37157, we have demonstrated that mitochondria accumulate large quantities of Ca2+ during a toxic glutamate stimulus and further that Ca2+ efflux from mitochondria contributes to the prolonged [Ca2+]i elevation after glutamate removal. We then used JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine+ ++ iodide), a ratiometric indicator of mitochondrial membrane potential (delta psi), to show that Ca2+ accumulation within the organelle dissipates delta psi. The abrupt loss of delta psi after glutamate stimulation did not occur in the presence of MK801 or in the absence of extracellular Ca2+. The mitochondrial depolarization was also cyclosporin A-sensitive, indicating a probable role for the permeability transition pore. Hence mitochondrial Ca2+ accumulation and the subsequent permeability transition may be a critical early event specific to the NMDA receptor-mediated excitotoxic cascade.

Topics: Animals; Benzimidazoles; Calcium; Carbocyanines; Carrier Proteins; Cells, Cultured; Clonazepam; Electrophysiology; Fluorescent Dyes; Fluorometry; Glutamic Acid; Indoles; Membrane Potentials; Mitochondria; Neurons; Neurotoxins; Prosencephalon; Rats; Receptors, Glutamate; Sodium-Calcium Exchanger; Thiazepines

Using the fluorescent membrane potential probe, 3,3'-dihexyl-oxacarbocyanine (DiOC6(3], we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K+), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positively-charged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux "pump" system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR "pump." How, or whether, plasma membrane potentials and the MDR efflux "pump" are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive

Topics: Animals; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Doxorubicin; Drug Resistance; Flow Cytometry; Indoles; Membrane Potentials; Mice; Potassium; Tumor Cells, Cultured; Verapamil