Catecholamine release from bovine adrenal medulla in response to maintained depolarization.
ABSTRACT Prolonged exposure of venous-perfused bovine adrenal glands to high K in the presence of external Ca produces a transient increase in catecholamine output that reaches a maximum after about 1 min and then declines with a half-time of about 1-2 min. 2. The time course of the transient secretory response to high K does not depend appreciably on the total catecholamine output which indicates that depletion of releasable catecholamine is unlikely to be responsible for the transient nature of the response. 3. Application of 3-6 mM-Ba stimulates secretion from a gland after many minutes exposure to high K, when catecholamine output has declined close to resting levels. This provides further evidence that depletion does not play a major role in the transient response and shows that maintained depolarization does not inhibit the secretory mechanism. 4. Exposure to high K solutions in which Ca has been replaced isomotically by Mg does not evoke any catecholamine output. Subsequent application of Ca always elicits some secretion although the size of this response to added Ca declines rapidly during exposure to Ca-free, high K solutions. The failure of the secretory response in these experiments is more rapid, and earlier in onset than the declining phase of the normal secretory response evoked in the presence of calcium. 5. Pre-treatment with Ca-free solutions of intermediate K content reduces the response to subsequent simultaneous application of high K and Ca. There is a roughly sigmoidal relation between the reduction in response and the logarithm of the K concentration used for pre-treatment. 6. Thin slices of bovine adrenal medulla show qualitatively similar responses on exposure to high K. Examination of the flourescent signal from slices dyed with the potential-sensitive dye DiS-C(3)-(5) suggests that maintained exposure to high K produces a stable depolarization. 7. The most likely explanation for these results is that K-depolarization first activates and subsequently inactivates a potential-sensitive Ca permeability channel. This inactivation is time and possibly potential dependent. 8. The effect of high K on calcium movements in medullary slices was examined. Exposure to 72 mM-K increases (45)Ca uptake, the increase being greatest during the first 10 min. The efflux of Ca is also increased on exposure to high K in the presence of Ca. The net Ca uptake in 72 mM-K is smaller than the tracer uptake of Ca. These findings indicate that K depolarization stimulates a Ca-Ca exchange process. They are also consistent with, but do not offer strong positive support for, the idea that K-depolarization first activates and subsequently inactivates Ca entry. 9. It is suggested that Ca inactivation might play a role in the modulation of neurosecretion and neurotransmitter release by changes in membrane potential.
- SourceAvailable from: Joaquín Jordán[show abstract] [hide abstract]
ABSTRACT: The molecular mechanisms involved in veratridine-induced chromaffin cell death have been explored. We have found that exposure to veratridine (30 microM, 1 h) produces a delayed cellular death that reaches 55% of the cells 24 h after veratridine exposure. This death has the features of apoptosis as DNA fragmentation can be observed. Calcium ions play an important role in veratridine-induced chromaffin cell death because the cell permeant Ca(2+) chelator BAPTA-AM and extracellular Ca(2+) removal completely prevented veratridine-induced toxicity. Following veratridine treatment, there is a decrease in mitochondrial function and an increase in superoxide anion production. Veratridine-induced increase in superoxide production was blocked by tetrodotoxin (TTX; 10 microM), extracellular Ca(2+) removal and the mitochondrial permeability transition pore blocker cyclosporine A (10 microM). Veratridine-induced death was prevented by different antioxidant treatments including catalase (100 IU ml(-1)), N-acetyl cysteine (100 microM), allopurinol (100 microM) or vitamin E (50 microM). Veratridine-induced DNA fragmentation was prevented by TTX (10 microM). Veratridine produced a time-dependent increase in caspase activity that was prevented by Ca(2+) removal and TTX (10 microM). In addition, calpain and caspases inhibitors partially prevented veratridine-induced death. These results indicate that chromaffin cells share with neurons the molecular machinery involved in apoptotic death and might be considered a good model to study neuronal death during neurodegeneration.British Journal of Pharmacology 09/2000; 130(7):1496-504. · 5.07 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Fast superfusion of electroporated bovine adrenal chromaffin cells with a K+ glutamate-based solution containing 50 nM free Ca2+ and 2 mM adenosine 5'-triphosphate, dipotassium salt (K2ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca2+ concentrations ([Ca2+]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca2+] (3-100 microM), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200-400 vesicles per cell. Pretreatment of cells with tyramine (10 microM for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca2+] in the range of 0.1-3 microM, the activation rate of secretion increased from 2.3 to 35.3 nA.s-1, reached a plateau between 3-30 microM and rose again from 100 to 1000 microM [Ca2+] to a maximum of 91.9 nA.s-1. In contrast, total secretion first increased (0.1-1 microM Ca2+), then plateaud (1-100 microM Ca2+) and subsequently decreased (100-1000 microM Ca2+). At 30 and 1000 microM extracellular [Ca2+] or [Ca2+]o, the activation rates of secretion from intact cells depolarised with 70 mM K+ were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 microM Ca2+) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 microA.s at 1000 microM Ca2+) than in electroporated cells (42.4 microA.s at 1 microM Ca2+). Separation of the time-courses of changes in intracellular [Ca2+] or [Ca2+]i and secretion in intact chromaffin cells depolarised with 70 mM K+ was demonstrated at different [Ca2+]o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca2+]i. In contrast, the decline of secretion was faster than the decline of the peak [Ca2+]i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca2+ by the rates of opening and closing of voltage-dependent Ca2+ channels of the L- and Q-subtypes, which control the local [Ca2+]i near to exocytotic sites.Pflügers Archiv - European Journal of Physiology 01/1996; 431(2):283-96. · 4.87 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The coupling between divalent cations and exocytosis of large dense-cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are "stimulus-coupled" to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus-coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20-50 sec. "Stimulus-decoupled" exocytosis is slow (approximately 25-40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5-12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.Journal of Neuroscience 03/1996; 16(4):1370-9. · 6.91 Impact Factor
J. Phy8iol. (1975), 253, pp. 593-620
With 13 text-figures
Printed in Great Britain
CATECHOLAMINE RELEASE FROM BOVINE ADRENAL
MEDULLA IN RESPONSE TO MAINTAINED DEPOLARIZATION
BY P. F. BAKER* AND T. J. RINK
From the Physiological Laboratory,
Downing Street, Cambridge (B2 3EG
(Received 13 June 1975)
1. Prolonged exposure of venous-perfused bovine adrenal glands to high
K in the presence of external Ca produces a transient increase in catechol-
amine output that reaches a maximum after about 1 min and then declines
with a half-time of about 1-2 min.
2. The time course of the transient secretary response to high K does
not depend appreciably on the total catecholamine output which indicates
that depletion of releasable catecholamine is unlikely to be responsible for
the transient nature of the response.
3. Application of 3-6 mM-Ba stimulates secretion from a gland after
many minutes exposure to high K, when catecholamine output has
declined close to resting levels. This provides further evidence that
depletion does not play a major role in the transient response and
shows that maintained depolarization does not inhibit the secretary
4. Exposure to high K solutions in which Ca has been replaced
isosmotically by Mg does not evoke any catecholamine output. Subse-
quent application of Ca always elicits some secretion although the size of
this response to added Ca declines rapidly during exposure to Ca-free,
high K solutions. The failure of the secretary response in these experi-
ments is more rapid, and earlier in onset than the declining phase of the
normal secretary response evoked in the presence of calcium.
5. Pre-treatment with Ca-free solutions of intermediate K content
reduces the response to subsequent simultaneous application of high K
and Ca. There is a roughly sigmoidal relation between the reduction in
response and the logarithm of the K concentration used for pre-treatment.
6. Thin slices of bovine adrenal medulla show qualitatively similar
responses on exposure to high K. Examination of the fluorescent signal
*Present address: Department of Physiology, King's College, Strand, London
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P. F. BAKER AND T. J. RINK
from slices dyed with the potential-sensitive dye DiS-C3-(5) suggests that
maintained exposure to high K produces a stable depolarization.
7. The most likely explanation for these results is that K-depolarization
first activates and subsequently inactivates a potential-sensitive Ca
permeability channel. This inactivation is time and possibly potential
8. The effect of high K on calcium movements in medullary slices was
examined. Exposure to 72 mM-K increases 45Ca uptake, the increase being
greatest during the first 10 min. The efflux of Ca is also increased on
exposure to high K in the presence of Ca. The net Ca uptake in 72 mM-K
is smaller than the tracer uptake of Ca. These findings indicate that K
depolarization stimulates a Ca-Ca exchange process. They are also con-
sistent with, but do not offer strong positive support for, the idea that K-
depolarization first activates and subsequently inactivates Ca entry.
9. It is suggested that Ca inactivation might play a role in the modula-
tion of neurosecretion and neurotransmitter release by changes in mem-
Catecholamine secretion from the adrenal medulla can be stimulated
by concentrations of potassium sufficient to depolarize chromaffin cell
membranes. This stimulation is dependent on the presence of external Ca
and is thought to involve entry of Ca into the cells, through a potential-
dependent Ca channel, the consequent rise in intracellular Ca triggering
catecholamine release (Douglas & Rubin, 1961).
Secretion is not maintained in the presence of high potassium (Douglas
& Rubin, 1963; Banks, Biggins, Bishop, Christian & Currie,
Possible explanations to account for this transient response include
depletion of releasable catecholamine, inactivation at reduced membrane
potentials of some component of the secretary system, and failure of high
K to maintain depolarization of chromaffin cells. In view of the evidence
that depolarization of squid axons produces only a transient entry of Ca
(Baker, Meves & Ridgway, 1973), an attractive possibility is that the
phasic release of catecholamine results from activation and subsequent
inactivation of Ca entry. The present experiments were designed to exa-
mine these possibilities. The general conclusion is that the phasic release of
catecholamine results from inactivation of some part of the secretary
mechanism, most probably the entry of Ca.
This work has been the subject of a brief communication to the Physio-
logical Society (Baker & Rink, 1974).
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INACTIVATION OF CATECHOLAMINE RELEASE
Materials. Bovine adrenal glands were obtained from a local abattoir. They were
removed from the animals as soon as possible, about 20 min after death, dissected
free from perinephric fat, infused through the adrenal vein with ice cold physiological
saline, and transported to the laboratory on ice.
Solutions. Two basic solutions were used, either bicarbonate buffered solution
134 mm-NaCl, 2 7 mm-KCl,
MgCl2 and 5-6 mm dextrose, or Tris (Tris-(hydroxymethyl)-amino-methane) buffered
1*8 mM-MgCI2and 5-6 mm dextrose. Bicarbonate buffered solutions were equilibrated
with 95% 02:5% C02, and Tris buffered solutions with 100% 02. The pH of all
solutions used was close to 7-4 at 370 C. Some perfusion experiments used the
bicarbonate buffered solutions, while the other perfusion experiments and all
experiments with slices used Tris buffered solutions. There was no discernible
difference in the responses of glands with the differently buffered solutions.
In solutions of raised K concentration, KCI replaced NaCl on an equimolar basis.
In all solutions, except those used for Ca tracer loading prior to efflux experiments,
the total divalent cation was 3-6 mx. Thus CaCl2 replaced MgCl2, or vice versa, on
an equimolar basis. In a few experiments 3-6 mM-BaCl2 was used. Bicarbonate
buffered solution was used when S04 replaced Cl. Here, SO4 replaced Cl and isotoni-
city was maintained with sucrose. When a high K, Cl-free solution was required,
K2SO4 isotonically replaced Na2SO4.
Perfu8ion. Glands were perfused retrogradely through the adrenal vein, as
described by Banks (1965). Perfusion solutions, gassed with the appropriate gas
mixture, were warmed by being passed through glass coils immersed in a water bath,
and led via a three-way tap to the preparation. Solutions could be switched rapidly
without interrupting the flow. The total effluent from the gland was collected and
2 ml. aliquots removed and acidified with 2 ml. 3-6% (w/v) HC104 for subsequent
When solutions were to be changed, the three-way tap was switched before the
collecting beakers were changed. The timing of the switch was such that the dead
space of the cannula and the vascular space of the medulla were just filled with the
new solution when the beakers were changed, so that the next collection period
started at the time the new solution reached the medullary tissue. The perfusion
rate was 9-12 ml./min and the temperature of the perfusing solutions measured
with a thermistor near the venous cannula was 37° C.
Glands were perfused for 30-40 min before the start of each experiment until
residual blood had been entirely washed from the vascular space of the gland and
the effluent was quite clear.
Preparation of medullary 8liew. Thin slices of bovine adrenal medulla were cut
using three 'Ever-ready' single-edged razor blades held parallel in a specially
designed brass holder. Slices were 150-250,umthick and weighed 30-60 mg. Any
small islands of cortical tissue were removed before the slices were used. Slices
were incubated for at least 60 min before experiments commenced.
Experimental procedure with licete. Slices from which catecholamine output or Ca
tracer efflux were to be measured were attached, with thin cotton thread, to the bent
ends of 3 mm polyethylene tubes. They were incubated in 2 ml. of solution, in poly-
ethylene vials, held in a water bath at 370 C. A steady stream of 100
through the support tube, producing both oxygenation and vigorous stirring. The
slices were incubated for a timed period and then transferred to fresh solution in
another vial by simply transferring the polyethylene tube from one vial to the
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P. F. BAKER AND T. J. RINK
next. The slices were attached to the concavity of the bent end of the tubes which
prevented damage to the slices during the transfer between vials. After an incubation
period, the solution in which the slice had been incubated was acidified with 2 ml.
3-6% (w/v) HC104 for subsequent catecholamine assay.
Ca tracer efflux experiments were performed with either 45Ca or 47Ca. Slices were
loaded with tracer Ca by exposing them to solutions containing 2-5 ,uCi/ml. of tracer
Ca and 0-2 mM 40Ca, for a 40 min period. In most experiments the slices were
alternately exposed, for 5 min periods to loading solutions containing 2-7 mM-KCl,
or 100 mm-KC1. In some experiments the loading was throughout in solutions
containing 100 mM-K. The Mg concentration in the loading solutions was only
1 mm. Following the loading period the slices were incubated in six changes of Ca
free solution, over a 48 min period. During this period extracellular tracer Ca is
removed from the slices. This washout period is not shown in the experimental
results. Following the washout period the slices were incubated for timed periods in
the appropriate experimental solutions, and the radioactivity released into the vials
measured. In some experiments 47Ca efflux and catecholamine output were measured
simultaneously. On these occasions after incubation the solution was acidified with
At the end of an efflux experiment the activity remaining in the slices was
measured. Slices loaded with 47Ca were placed in an empty vial for counting. Those
loaded with 45Ca were placed in a glass scintillation vial, dissolved in 0-1 ml. con-
centrated HNO3 and evaporated to dryness. The residue was decolorized with H202,
and again evaporated to dryness, dissolved in 0-2 ml. distilled water and scintilla-
tion solution added.
The Ca tracer efflux was calculated as the fraction of tracer Ca lost per minute.
47Ca was used in some experiments because of availability and ease of counting.
This isotope decays with a half-life of 4-3 days to 47Sc which is also a gamma emitter.
This could possibly interfere with measurement of Ca movements but in these
experiments there seemed to be little difference between results obtained with 45Ca
and those with 47Ca. The results of experiments using 47Ca have been reported, but
only where essentially similar results have been obtained with 4Ca.
45Ca uptake. 45Ca only was used for tracer uptake experiments. Many slices were
cut from several glands and divided into groups of six to eight. They were incubated
in polyethylene beakers, held in a water-bath at 370 C. The incubation solutions
were bubbled with 100%02 which both oxygenated and stirred them. After about
90 min equilibration in several changes of solution the slices were transferred to the
appropriate pre-incubation medium for a 10 min period. This might be high or low K
solution. Each group of slices was subsequently transferred to the appropriate 45Ca
uptake solution for a further 10 min period. The 45Ca content of the uptake solutions
was about 0-2,uCi/ml.Asample ofeach solution wasthen taken during the experiment
for the measurement of its specific activity, so that Ca uptake could be expressed
in absolute terms. The composition of the various uptake solutions is described in
the results. After the uptake period, the slices were incubated in six changes of
standard, 3-6 mM-Ca solution at room temperature, over a 36 min period, in order to
remove extracellular 45Ca. They were then blotted, weighed, and prepared for
scintillation counting in the manner described for slices at the end of 45Ca efflux
The loss of 45Ca from slices incubated in standard 3-6 mM-Ca solution at room
temperature shows two main components. There is a rapid early component with a
half-time of about 3 min which probably represents the washout of extracellular
4"Ca while the slow component which has a half-time of about 70 min probably
represents 45Ca efflux from the cells. The 36 min washout period used should be
sufficient to remove the extracellular 45Ca but probably also removes some 30 % of
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INACTIVATION OF CATECHOLAMINE RELEASE
the intracellular C50a. The measured 45a uptakes will thus be underestimated by
about 30 %. Since all the slices underwent the same washout procedure it is assumed
that the measured relative "Ca uptake in the different groups is a fair reflexion of
the actual relative "Ca uptake. 45Ca uptake has been expressed as Emole (kg wet
Total Ca content. After incubation slices were passed through five changes of ice
cold standard Ca-free (3-6 mm-Mg) solution over a 15 min period. Slices were then
blotted, weighed and dissolved in 0-2 ml. concentrated HN03. The resulting solution
was made up to 4 ml. with distilled water and the Ca concentration measured by
atomic absorption spectroscopy.
Measurement of membrane potential using fluorescent dyes. 0-05 ml. of a saturated
ethanolic solution of the dyeDiS-C,-(5)(Sims, Waggoner, Wang & Hoffman, 1974)
was added to 10 ml. standard solution in a polyethylene beaker, at 370 C. Slices were
incubated in this solution for about 15 min until they were light green in colour.
They were then incubated for about 50 min in dye free solution. A dyed slice was
placed in between two thin sheets of black Perspex in which holes had been cut so
that only the edge of the slice was held between the pieces of Perspex while the
largest part of the slice, on both surfaces, was exposed. This assembly just fitted
across the corners of a fluorescence cuvette so that the slice was firmly held in the
cuvette at 450 to the sides. The fluorescence was measured in a Perkin Elmer 203
Fluorescence Spectrophotometer. A flow system allowed solutions to be changed
without removing the preparation from the spectrophotometer. The level of the
solution in the cuvette was held constant by a suction tube, while the temperature,
which was maintained at 370 C, was monitored with a thermistor in the solution. The
slice was placed so that the incident light path was at 45°, and only scattered light,
passing through the dyed slice, could reach the photomultiplier. The emission
wave-length was 620 nm, and the analyzing wave-length 670 nm. Standard solution
was normally flowed through the cell for about 30 min to establish a base line before
the solution flowing into the cell was changed, without interrupting the flow. The
fluorescent signal was recorded on a pen-recorder.
Assay. Catecholamine was measured in a Perkin Elmer 203 fluorescence spectro-
photometer by the trihydroxyindole method. Total catecholamine was measured
against an adrenaline standard. In a few experiments in which adrenaline and
noradrenaline were separately estimated the adrenaline/noradrenaline ratio was
fairly constant in the effluent from both resting and stimulated glands.
45Ca was counted on a Nuclear Chicago scintillation counter. A channels ratio
method was used to check for errors due to quenching. 47Ca was counted on a Nuclear
Enterprises gamma counter. The counting of samples was done rapidly enough to
render unnecessary a correction for radioactive decay. Ca concentration in samples
was measured on a Unicam SP 90B atomic absorption spectrophotometer.
Chemicals. Tris (Trizma base) was obtained from Sigma Chemical Co, adrenaline
acid tartrate from B.D.H. Ltd and the radioactive isotopes from Radiochemicals,
Amersham. Analar reagents were used where available. The DiS-C3-(5) was a gift
from Dr Brian Salzburg.
Analysis of results. Quantitative comparison ofthe secretary responses ofperfused
glands stimulated under different test conditions was complicated by two factors.
(1) There was variation between glands in the size of responses. This was overcome
by comparing test and control responses in the same gland and expressing the
response under test conditions as a percentage of that under control conditions.
(2) The response of any one gland to successive stimulation declined progressively
throughout the course ofan experiment. This is a feature of the response ofperfused
glands, as has been previously noted (Douglas & Rubin, 1961). To meet this difficulty
each test response was bracketed by control responses. The simplest way to
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