Calcium gradients and buffers in bovine chromaffin cells.

Abstract

SUMMARY1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2" indicator, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells.2. Depolarization with a patch pipette produced radial gradients of Ca21 within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca21 channels were closed.3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2 .4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected.5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99 % of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 /LMCa2+ inside the cell.6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (04 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions ratiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.MS 9349

BDNF enhances dendritic Ca2+ signals evoked by coincident EPSPs and back-propagating action potentials in CA1 pyramidal neurons

“…Custom-written software was used to perform synchronized electrical and optical recordings with control of the amplifier, stimulator, and mono-chromator (courtesy of Dr. Takafumi Inoue, The University of Tokyo, Japan), as described (Pozzo-Miller et al, 1999). Intracellular Ca 2+ concentration was estimated from bisfura-2 fluorescence images using the isosbestic method (Neher and Augustine, 1992). Image frames at 357 nm excitation (Ca 2+ -insensitive wavelength calibrated in our system) were acquired before and after a continuous sequence of 380 nm excitation frames to generate pixel-by-pixel ratio images and time course plots.…”

Short-Term Presynaptic Plasticity

“…After calcium channels close, spatial gradients collapse as calcium diffuses and binds to calcium-binding proteins within the presynaptic bouton (Fogelson and Zucker 1985;Simon and Llinas 1985). The remaining calcium, known as residual calcium (Ca res ), is then gradually extruded from the presynaptic bouton (Neher and Augustine 1992;Tank et al 1995). Ca res levels are much lower (hundreds of nanomolar) and longerlived (hundreds of milliseconds to seconds) than those of Ca local , and play an important role in short-term plasticity.…”

Mitochondria and chromaffin cell function

“…It is a general observation that calcium binds avidly to endogenous Ca 2+ -binding proteins in all the intracellular compartments including the cytosol, so that the total calcium concentration ([ T Ca]) is always much larger than the ionised one ([Ca 2+ 2+ ] or calcium-binding capacity [40] quantifies this buffering effect. Differences in buffering power among different compartments do not generate [Ca 2+ ] gradients but differences in their [ T Ca].…”

“…κ S values 1,000 have been reported for myocytes [58] and various neuronal types [1,23,46,54]. On the other hand, values of 40-100 have been reported for rat chromaffin cells [11,29] and bovine chromaffin cells [40,60,63].…”

“…Cytosolic Ca 2+ buffering and diffusion in bovine chromaffin cells has been extensively studied by Neher and coworkers [40,60,63]. The cytosol has a total binding capacity of 4 mM, and the endogenous Ca 2+ buffer is fast (association rate010 −8 M −1 s −1 ), poorly mobile, and with low affinity for Ca 2+ (dissociation constant0100 μM).…”