No abstract

A growing body of evidence proposes that glial cells have the potential to play a role as modulators of neuronal activity and synaptic transmission by releasing the neurotransmitter glutamate (Arague et al., 1999). We explore the spatial nature of glutamate release from astrocytes with an enzyme-linked assay system and CCD imaging technology. In the presence of glutamate, L-glutamic dehydrogenase (GDH) reduces NAD(+) to NADH, a product that fluoresces when excited with UV light. Theoretically, provided that GDH and NAD(+) are present in the bathing saline, the release of glutamate from stimulated astrocytes can be optically detected by monitoring the accumulation of NADH. Indeed, stimuli that induce a wave of elevated calcium among astrocytes produced a corresponding spread of extracellular NADH fluorescence. Treatment of cultures either with thapsigargin, to deplete internal calcium stores, or with the membrane-permeant calcium chelator BAPTA AM significantly decreased the accumulation of NADH, demonstrating that this fluorometric assay effectively monitors calcium-dependent glutamate release. With a temporal resolution of 500 msec and spatial resolution of approximately 20 micrometer, discrete regions of glutamate release were not reliably resolved. The wave of glutamate release that underlies the NADH fluorescence propagated at an average speed of approximately 26 micrometer/sec, correlating with the rate of calcium wave progression (10-30 micrometer/sec), and caused a localized accumulation of glutamate in the range of 1-100 microM. Further analysis of the fluorescence accumulation clearly demonstrated that glutamate is released in a regenerative manner, with subsequent cells that are involved in the calcium wave releasing additional glutamate.

We have inflated patch-clamped mast cells by 3.8 +/- 1.6 times their volume by applying a hydrostatic pressure of 5-15 cm H2O to the interior of the patch pipette. Inflation did not cause changes in the cell membrane conductance and caused only a small reversible change in the cell membrane capacitance (36 +/- 5 fF/cm H2O). The specific cell membrane capacitance of inflated cells was found to be 0.5 microF/cm2. High-resolution capacitance recordings showed that inflation reduced the frequency of exocytotic fusion events by approximately 70-fold, with the remaining fusion events showing an unusual time course. Shortly after the pressure was returned to 0 cm H2O, mast cells regained their normal size and appearance and degranulated completely, even after remaining inflated for up to 60 min. We interpret these observations as an indication that inflated mast cells reversibly disassemble the structures that regulate exocytotic fusion. Upon returning to its normal size, the cell cytosol reassembles the fusion pore scaffolds and allows exocytosis to proceed, suggesting that exocytotic fusion does not require soluble proteins. Reassembly of the fusion pore can be prevented by inflating the cells with solutions containing the protease pronase, which completely blocked exocytosis. We also interpret these results as evidence that the activity of the fusion pore is sensitive to the tension of the plasma membrane.

The nuclear envelope represents a structural and functional barrier between cytoplasm and nucleoplasm. Small molecules and solutes passively cross the nuclear envelope, whereas the transport of large proteins and RNA requires metabolic energy. Using in situ Xenopus oocyte nuclei, we characterized ATP-dependent ionic permeabilities on the external surface of the envelope. The presence, but not necessarily the hydrolysis, of ATP is crucial to maintaining the channels in an open state. Localization of the ionic channels is still unclear. From morphologic and current kinetics data, we suggest a relation between the ionic channels and the nuclear pores. We try, in this way, to explain the apparent contradiction between the presence of ion-selective channels in parallel with large aqueous pores on the nuclear envelope. Under this hypothesis, variations in the metabolic energy content of the cytoplasm would induce nucleocytoplasmic passive exchanges. The distribution and movement of charged particles across the nuclear envelope may influence many cytoplasmic functions. Regulation of the current by ATP could play an important role in hormonal stimulation, divalent ion permeation into the nucleus, and cell cycle mechanisms.

The rat basophilic cell line RBL-1 is known to express high levels of the Ca 2؉ current activated by store depletion, known as Ca 2؉ release-activated Ca 2؉ current (I CRAC ), the main Ca 2؉ influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca 2؉ influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between I CRAC and cell metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces I CRAC induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of I CRAC , since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca 2؉ buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis. Moreover, the inhibition is specific for ADP and is partially mimicked by ADP␤S and AMP, but not by GDP or GTP.