Rat basophilic leukaemia cells, like mast cells from which they are derived, have surface Fc epsilon receptors that trigger secretion of inflammatory mediators when crosslinked. Both GTP-binding proteins and a rise in cytosolic calcium concentration ([Ca2+]i) are implicated in the secretory mechanism. Here we use a video-imaging technique to report that transient rises in [Ca2+]i initiated in an individual cell can spread from cell to cell in a wave-like pattern by means of a secreted intermediate, in the absence of gap-junctional communication. We find that the leukaemia cells, peritoneal mast cells and mucosal mast cells have cell-surface P2-type purinergic receptors that can trigger similar [Ca2+]i transients. We provide evidence that ATP is rapidly released, and that it can amplify [Ca2+]i signals and initial secretory responses during antigen-stimulation of rat basophilic leukaemia cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP‐gamma‐S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura‐2) and measurement of the Ca2(+)‐dependent Cl‐ current (patch‐clamp whole‐cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1‐0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)‐dependent Cl‐ current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl‐ current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP‐gamma‐S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl‐ current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

SUMMARY1. Low-voltage-activated Ca2+ channels which produce a transient inward current were studied in neurones freshly isolated from the ventromedial hypothalamic region of the rat. Membrane currents were recorded using a suction-pipette technique which allows for internal perfusion under a single-electrode voltage clamp. A concentrationjump technique was also used for rapid drug application.2. In most cells superfused with 10 mM-Ca2+, a transient inward Ca2+ current was evoked by a step depolarization to potentials more positive than -65 mV from a holding potential of -100 mV. Such a low-threshold Ca2+ current could easily be separated from a high-threshold, steady type of Ca2+ current by selecting the holding and test potential levels, as well as by resistance to the wash-out during cell dialysis.3. Activation and inactivation processes of the low-threshold Ca2+ current were highly potential dependent at 20-22 'C. For a test potential change from -60 to + 20 mV, the time to peak of the current decreased from 45 to 9 ms, and the time constant of the current decay decreased from 90 to 40 ms. The steady-state inactivation occurred at very negative potentials, reaching a 50 % level at -93 mV. Recovery from inactivation showed a time constant between 2-63 and 0-94 s for a potential change from -80 to -120 mV.4

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drng-efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug-sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDRl-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by C1-channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl-conductance (gc1) were indistinguishable between control and corresponding multidrug-resistant cells: gcl reached 0.96 _+ 0.31 (n = 14) and 0.83 -0.31 nS/pF (mean -+ SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 + 0.20 (n = 9) and 0.37 + 0.22 nS/pF (n --7) in 8226 and 8226/Dox40 cells, respectively, gcl exhibited moderate outward rectification in symmetrical Cl-solutions, with a rectification ratio of 1.4 at -+ 50 mV. C1-channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored C1--channel opening in intact drug-sensitive and -resistant cells, gcb measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 p~M at +40 mV), NPPB (Ka ~ 30 ~M), and tamoxifen (complete and irreversible block ~ 10 IzM). None of these blockers inhibited R123 effiux. NPPB accelerated R123 efflux, an effect that was mimicked by CCCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 I~M); 10 p~M left gcl unaltered. Induction ofgcl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling-activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.