Functional and morphological characterization of isolated bovine adrenal medullary cells.

Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% collagenase in Ca++-free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 X 10(6) cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min. Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenaline-containing and (b) noradrenaline-containing cells. Biochemical analysis showed that the cells contained catecholamines and dopamine-beta-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca++ have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.

Cholinergic synaptic vesicles from Torpedo marmorata contain an atractyloside-binding protein related to the mitochondrial ADP/ATP carrier.

Atractyloside is known to bind to the ADP/ATP translocase of the inner mitochondrial membrane, a complex formed by two basic protein subunits of relative molecular mass around 30 000. We found that synaptic vesicles from the electric organ of Torpedo marmorata, which store acetylcholine and ATP, bind atractyloside as well. Similarly to mitochondria, a protein-atractyloside complex could be solubilized from vesicle membranes with Triton X-100. Characterization of the complex by gel filtration, isoelectric focusing and gel electrophoresis revealed that atractyloside was bound to protein V11, earlier described as a major vesicle membrane component with a relative molecular mass around 34 000 and a basic isoelectric point. Since earlier experiments have already shown that uptake of ATP into isolated vesicles in vitro is inhibited by atractyloside, we can conclude now that V11 constitutes the nucleotide carrier of this secretory organelle. The structural and functional relationship of the mitochondrial and vesicular nucleotide translocases suggest a common evolutionary origin.

A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine.

1. Bovine chromaffin cells were enzymatically isolated and kept in short term tissue culture. Their electrical properties were studied using recent advances of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). 2. When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached configuration') current wave forms due to intracellular action potentials could be observed. The frequency of the wave forms was altered by changing the pipette potential. When acetylcholine was present in the pipette solution, acetylcholine-induced single channel currents were evident in the patch recording. Action potential wave forms were then often seen to follow acetycholine-induced single channel currents. 3. In the cell-attached configuration, large single channel current events did not resemble square pulses but showed exponential relaxations with time constants of the order of 50 ms. 4. After rupture of the patch of membrane, the pipette--cell seal remained stable ('whole-cell recording', Hamill et al. 1981). Chromaffin cells were found to have a resting potential of -50 to -80 mV, and an input resistance around 5 G omega. The high cell resistance accounts for the relaxing currents evident in the cell-attached configuration. 5. In the best cases, the effective time constant of the voltage clamp in the whole-cell recording mode was 15 microseconds. Exchange of small ions such as Na+ ions between pipette and cell interior solutions was then complete within 15 s. 6. Acetylcholine-induced currents were obtained at various acetylcholine concentrations. Single acetylcholine-induced channels had a slope conductance of 44 pS between -100 and -55 mV, and a mean duration of 27 ms at -80 mV (at room temperature).

Sodium and calcium channels in bovine chromaffin cells.

1. Inward currents in chromaffin cells were studied with the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The intracellular solution contained 120 mM-Cs(+) and 20 mM-tetraethylammonium (TEA(+)). Na(+) currents were studied after blockade of Ca(2+) channels with 1 mM-Co(2+) applied externally. Ca(2+) currents were recorded after eliminating Na(+) currents with tetrodotoxin (TTX). The current recordings were obtained in cell-attached, outside-out and whole-cell recording configurations (Hamill et al. 1981).2. Single channel measurements gave an elementary current amplitude of 1 pA at -10 mV for Na(+) channels. This amplitude increased with hyperpolarization between -10 and -40 mV, but did not vary significantly between -40 and -70 mV.3. The mean Na(+) channel open time was 1 ms at -30 mV. This open time decreased both with depolarization and hyperpolarization. Its value was close to the time constant of inactivation, tau(h), above -20 mV.4. Ensemble fluctuation analysis of Na(+) currents gave results consistent with those of single channel measurements. Noise power spectra obtained between -35 mV and 0 mV could be fitted with a single Lorentzian. A range of Na(+) channel densities of 1.5-10 channels per mum(2) was calculated.5. Cell-attached single Ca(2+) channel recordings were obtained in isotonic BaCl(2) solution. The single channel amplitude was 0.9 pA at -5 mV, and it became smaller for positive potential values.6. At -5 mV, single Ba(2+) currents appeared as bursts of 1.9 ms mean duration containing on the average 0.6 short gaps. The burst duration was larger at positive potentials.7. Ensemble fluctuation analysis of Ca(2+) channels was performed on whole-cell recordings in external solutions containing isotonic BaCl(2) or external Ca(2+) (Ca(o)) concentrations of 1 and 5 mM. The unit amplitude calculated in the former case was similar to that obtained in single channel measurements.8. Noise power spectra of Ca(2+) or Ba(2+) currents could be fitted by the sum of two, but not one, Lorentzian components.9. Tail currents could be fitted by the sum of two exponential components. The corresponding time constants had values close to those obtained with noise analysis.10. The rising phase of Ca(2+) and Ba(2+) currents was sigmoid. It could be fitted by the sum of three exponentials. The time constant of the largest amplitude component, tau(1), was similar to the time constants of the slow component observed in noise and tail experiments. This time constant also corresponded to the burst duration obtained in single channel measurements.11. The value of tau(1) was larger in 5 mM-Ca(o) and in isotonic Ba(2+) than in 5 mM-Ba(o). Thus, the kinetic properties of Ca(2+) channels depend on the nature and concentration of the permeating ion.12. A simple kinetic scheme is proposed to model the activation pathway of Ca(2+) channels.13. Currents in 1 mM-Ca(o) and 5 mM-Ca(o) showed clear reversals around +53 mV and +64 mV respectively. The outward currents observed above these potentials are most probably due to Cs(+) ions flowing through Ca(2+) channels.14. The instantaneous current-voltage relation was obtained from tail current data in the range -70 to +100 mV in 5 mM-Ca(o). The resulting curve displayed an inflexion point around the reversal potential.15. Very little inactivation of Ca(2+) currents was observed. However, a slow current decline was observed in some cells above +10 mV.16. Conditioning prepulses to positive potentials had potentiating or depressing effects on Ca(2+) currents depending on whether the test pulse lay below or above the maximal current potential. The potentiating effect may be linked to the slowest component of the current rise observed below +10 mV. The depressing effect may be related to the slow decline obtained above +10 mV.17. Analysis of ensemble variance and of tail current amplitudes suggested that the opening probability of Ca(2+) channels was at least 0.9 above +40 mV.18. A slow rundown of Ca(2+) currents was observed in whole-cell recordings. The speed of the rundown was dependent on intracellular Ca(2+) concentration. The rundown was apparently due to a progressive elimination of the channels available for activation.19. The density of Ca(2+) channels (before rundown) was estimated at 5-15/mum(2).20. In cell-attached experiments, inward current channels were often seen to follow action potentials. These events did not appear to be the usual Na(+) and Ca(2+) currents. They were probably due to cation influx of either Na(+) or Ba(2+), depending on the pipette solution, through Ca(2+)-dependent channels. Voltage-independent single channel activity observed in whole-cell and outside-out recordings may be due to the same channels.

Immunochemical comparison of synaptic plasma membrane and synaptic vesicle membrane antigens.

A synaptic vesicle fraction and a synaptic plasma membrane fraction obtained after subfractionation of synaptosomes from chick forebrain have been used to produce antisera in rabbits. Immunofluorescence histology with the two antisera revealed that they reacted strongly with synaptic terminal regions present in the chick forebrain, cerebellum and spinal cord. In addition, the synaptic plasma membrane antiserum (but not the synaptic vesicle antiserum) reacted with preterminal axons in the cerebellum and spinal cord. Comparison of the two antisera by two-dimensional immunoelectrophoresis, revealed the presence of common antigens in the synaptosomal vesicle and plasma membrane fractions. Incubation of synaptosomes in vitro with the synaptosomal vesicle antiserum and complement produced a dose-dependent inhibition of synaptosome swelling up to a maximum of 55% of that obtained with the synaptosomal plasma membrane antiserum. The results of this test are consistent with the hypothesis that some synaptosomal vesicle antigens may be present also in the synaptosomal plasma membrane and imply that they face the external surface of the synaptosomes. The fate of vesicle membrane components in synaptosomal plasma membranes is not known. The possibility is discussed that they may be recycled locally by a mechanism similar to that proposed by Heuser and Reese (1973) for re-use of synaptic vesicle membranes at the neuromuscular junction.