Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and ω-conotoxin-insensitive pathways

03064522/89 %3.00+ 0.08 Pergamon Press plc 0 1989 IBRO

~~~rosei~n~e Vol. 29, No. 3, pp. 735-747, 1989 Printed in Great Britain

VOLTAGE-SENSITIVE CALCIUM FLUX INTO BOVINE CHROMAFFIN CELLS OCCURS THROUGH DIHYDROPYRIDINE-SENSITIVE AND DIHYDROPYRIDINEAND o -CONOTOXIN-INSENSITIVE PATHWAYS L. M. ROSARIO,*E~B. SORIA,? G. FEUERSTEIN$ and H. B. POLLARD* *Laboratory of Cell Biology and Genetics, National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892, U.S.A. tDepartment of Physiology, School of Medicine, University of Alicante, Alicante, Spain SDepartment of Neurology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, U.S.A. Abstract-The fluorescent Ca*+ indicator FURA- was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (l&65 mM) gave rise to a very rapid increase in intracellular free Ca2+ concentration, which subsequently decayed slowly towards a “plateau”. The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, - 23 mV). A dihydropyridine calcium channel agonist [( + 1202-791, I p M] raised int~~llular free Ca2+ concentration further in the presence of 30 mEAK+, and it enhanced the initial intracellular Ca*+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the L-type. Several dihydropyridine calcium channel antagonists [( -)202-791, nifedipine, nitrendipine; l-5 PM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, w-conotoxin GVIA (l-10 PM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca*+ concentration only slightly at 55 mM K+. Further, the dihydropy~dine-insensitive component of the int~~llular Ca2* signal was also insensitive to w-conotoxin, which was otherwise quite active in a central nervous rat in uiuo preparation. Gd’+ (40 PM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ concentration to sub-nanomolar levels, in the presence of high K+. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-de~ndent calcium channels, only one of which is likely to be the L-type channel. We also propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.

Adrenal medulla chromaffin cells secrete catecholamines in response to the neurotransmitter acetyl-

choline (ACh) and other choline@ agonists. The secretory response has an absolute requirement for extracellular calcium,12 indicating that Ca*+ influx is the primary trigger of the secretion cascade that finally leads to exocytotic release of the catecholamines. While the mechanisms by which Ca2+ trans-

$Present address: Department of Physiology, School of Medicine, University of Alicante, Alicante, Spain. Abbreoiutions: ACh, acetylcholine; BSA, bovine serum albumin; [Ca2+&, intracellular free calcium ion concentration; [Ca*+I,, extracellular calcium con~ntration; DHP, 1,~ihydropy~dine; DMSO, dimethyl sulfoxide; EGTA, ethyleneglycolbis-(B-aminoethylether)N,N,N’, N’-tetra-acetic acid; FURA-Z/AM, acetoxymethyl ester of FURA-2; HEPES, N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid; Quin-Z/AM, acetoxymethyl ester of Quin-2; TRIS, tris(hydroxymethyl)-aminomethane.

location occurs across the chromaffin cell membrane have been the subject of intense research, some important issues still await ctarification. For example, while the existence and operation of voltagedependent Ca channels has been relatively well documented in the chromaffin cell, *J~***J~ their relative importance compared to other transporters of Ca*+ ions (voltage-dependent Na channel(s), ACh ~eptor-operate ion channel) remains to be fully established.” According to a generally accepted physiologic “flow diagram” of ionic events leading to Ca2+ translocation across the chromaffin cell plasma membrane, ACh opens up a non-specific receptoroperated cation channel, which in turn depolarizes the membrane and activates voltage-de~ndent calcium channels.25 This concept emphasizes the importance of membrane potential changes and calcium channel activation in secretion. Depolarization of chromaffin cells with high K’ has often been used to mimick the depolarizing effects 73s

736

L. M. Ros~~io V/
of cholinergic stimulation. High K’ is known to elicit a dose-dependent secretion of catecholamines, a pracess which appears to be preceded by stimulation of Ca2+ uptake. ‘J’.~ This leads to a rapid increase in cytoplasmic free Ca2+ levels,8*27.39 which is thought to represent the first intracellular transducing signal for exocytosis. However, in spite of the important mechanistic implications of this concept, the depolarization-related [Ca*+J signalling process remains poorly characterized in the chromathn ceil. For example, while in one study2’ [Cal+ Ii has been shown to first increase and then decay in response to Ki stimulation, other authors have reported sustained [Ca2+‘fi changes.8.39.40The later data would be in conflict with recent studies2 showing that a peak increase in 45Ca2+ uptake is followed by a rapid and profound jnactivation of the uptake. By inference the later work has been taken to be indicative of calcium channel inactivation. In addition to the dihydropyridine (DHP) blockable current &-type), other types of Ca*+ currents including N- and T-type. currents have been described in neurons and other tissues.32.33Neuronal Land N-type channels have been ,shown to be blocked by the cone snail toxin w-conotoxin.3’-33,37 However, the limited analysis of calcium flux mechanisms in chromaffin cells using pha~acoIo~c criteria have generated, at best, conflicting conclusions. Whereas some authors have claimed extensive blockade of nicotine- and high K+-stimulated secretion by DHP blockers (e.g. nitrendipine),“ others have reported an impo~ant component of DHP-insensitive secretion, amounting to as much as 30%.5 In an attempt to gain further insight into depolarization-related intracellular Ca’+ movements in chromaffin cells, we have utilized in this study a recently developed intracellular fluorescent Ca’+ indicator, FURA-2,” to determine whether different pharmacologic classes3’.“’ of voltage-dependent calcium channels might be present and operative in the chromaffin cell membrane. EXPERIMENTAL

PROCEDURES

Ceil preparation and culture

Bovine adrenal glands were obtained from the local slaughterhouse and kept on ice for 45 min before use. The glands were then Perfused three times at 37°C with Ca*+and Mg2+ -free Locke’s solution containing fti): 154 NaCl, 5.6 KCI, 5 NaHCO,, 5.6 glucose and 5 HEPES (pH 7.4). The medullary tissue was dissociated by three successive perfusions (15 min each) with sterile Ca2+ - and Mg2+ -free Locke’s solution containing 0.5% BSA &Sigma, type IV) and OS%, 0.1% and 0.1% collagenase (Sigma, type VI), respectively, at 37°C. The medullae were then removed, minced in Locke’s medium and filtered through nylon gauze. Filtered cells were pelleted and washed twice in Locke’s medium. Chromaffin cells were further purified on a Percoll gradient.29 The final cell suspension typically contained about 80% chromaflin cells, as indicated by Neutral Red staining, with the remaining cells being mostly cortical and endothelial cells. Cells were cultured in a 1: 1 mixture of Dutbecco’s modified Eagle’s medium/Ham’s F-12 medium buffered with 15 mM HEPES and 21 mM

NaHCO,,

and supplemented

with 5% heat-macttvated

ictal

bovine serum (Sigma), penicillin (100 units;mi), sireptomycin {l~~g/ml~ and Fungizone (250 ngjmi). Cells weir cultured at a density of I .5 x lo* cells/ml in tissue culture flasks (Costar 3075). Bufler solutions

The following modified Krebs’ solutions were used throughout the experiments. Solution A (mM): 130NaCl. 5 KCI, 2 CaCl,, I MgCl,, 15 Na-HEPES, 10 glucose @H 7.35). Solution B (“Ca’+-free”): same as A, but with no CaCl, added. Solution C (Car+ -free, EGTA-containing): solution B supplemented with 2 mM EGTA. In some experiments, an Na* -free solution with the following composition (mM) was also used: 140 N-methy o-glucaminate chloride, 2 CaCl,, 1 MgCt,, 10 HEPES (acid), titrated to pH 7.4 with KOH ffinal K’. concentration 7.3 mM). Chromaffia cells in suspension, harvested from tissue culture flasks, were loaded with FURA- by incubation with the parent acetoxymethyl ester FURA-‘L/AM (Molecular Probes, Eugene, OR). Typically, cells (c. lO*/ml) were loaded in the oriainal culture medium with FURA-Z/AM (5 PM) for 60 mil at 37°C. FURA-2/AM was added from a 1 mM stock solution in DMSO. The parent compound diffuses across the membrane and is hydrolysed by internal esterases in the cytoplasm, releasing the poorly permeant FURA-2, a Ca*+ indicator.‘* The extracellular probe was removed by washing the cells twice, first with medium containing 1% BSA (Calbiochem) and then with BSA-free medium. The final peilet of loaded cells was resuspended in c. 0.7 ml medium (Solution A), and stored on ice to minimize FURA- leakage. This treatment did not affect the ability of chromailin cells to secrete catecholamines in response to high K* or nic0tine.l’ Continuous monitoring of FURAfluorescence was carried out in a SPEX Fluorolog spectrofluorometer (Spex Industries, Edison, NJ), equipped with a digital plotter and a microprocessor (DMlB). Excitation and emission wavelengths were set at 340 and 510 am, and I-2-am slits were used, respectively. A 434-nm cut-off long pass filter was used across the emission path to reduce scattered light. For the containing approximately recording small aliquots 3--S x lo6 FURA-2-loaded cells were transferred into acrylic cuvettes containing 2.5 ml medium at room temperature (c. 25°C). Cells in the cuvette were gently stirred and allowed to reequilibrate for c. 5 min. by which time the signal had become stable. Calibration offluorescence in terms of [Ca2+], was carried out at the end of each experiment by disrupting the cell membranes with the non-ionic detergent C,,E, (1 mM; Calbiochem), thus releasing the probe into the medium. This allowed the maximum (saturating) fluorescence of FURA(F,,,) to be recorded ‘(Fig. la,b). Next, a small aliquot (50~1) of an EGTA/TRIS mixture (0.2M EGTAjl M TRIS, pH 10.2) was added to the cuvette to reduce the Ca*+ concentration to sub-Mnomolar levels, thus allowing the minimum FURA- fluorescence (F.,,,,) to be recorded (Fig. la, b). Fluorescence intensities (F) were automatically converted point by point into ]cazfli values using the calibration equation for single wavelength excitation measurements: 1

[Caa+], = K,(F - F,,,)/(F,,,

= FL

.

.._

(it

where & is the association constant of the CaZ’ JFURA-2 complex, which was taken to be 224nM.l’ Addition of Co2+ (1 mM) to FURA-a-loaded cells resulted in a strong guorescence quenching. On the other hand, addition of Cd*+ (1 mM) rapidly and potently enhanced the fluorescence. Mgr+ (20mM) did not affect the fluorescence when added to intact FURA-2-loaded cells, but it impaired the calibration procedure by competing with

Dihydropyridine-insensitive

calcium entry into chromaffin cells

Ca2” for the (2~~M) fluorescence trations. For 8) calibration out.

Ca*+ JFURA-2 complex. Both La3+ and Gd’+ were found to quench FURA(acid) in the presence of ~turating Ca*+ concenthis reason, in experiments with Gd3+ (see Fig. of fluorescence in [CaZ+&units was not carried

Presentation

of results

records was based on a numeric aIgorit~ proposed by Savitzky and GoIay.42

737 originally

The dihydropyridine agonist and antagonist (+) and (-) 202-791 were a gift from Drs D. Romer and E. Rissi, Sandoz Ltd, Basle, Switzerland. Nitrendipine was a gift from Dr A. Animal preparation and experimental protocol for the study Garcia, University of Alicante, Alicante, Spain. The of effects of o-conotoxin administration in vivo o-conotoxin GVIA was from Peninsula Laboratories, Inc., Belmont, CA. Gadolinium chloride was from Alfa Products, Male Sprague-Dawley rats were anesthetized with ketamine/acepromazine (100/l mg/kg respectively, i.m.) and Morton Thiokol, Inc., Danvers, MA. All other reagents mounted on a stereotaxic device (DKI, CA). After a midwere from Sigma Chemical Co., St Louis, MO. Concenscalp incision the bregma was identified and the bone trated stock solutions of (+) and (-) 202-791, nifedipine exposed at L = 0.8, AP = - 1.2,in referencc to the bregma. and nitrendipine were prepared in DMSO, sampled in small A guide cannula was then implanted through a hole drilled aliquots, and kept at -70°C prior to use. Occasionally, in the skull and fixed with instant glue (Eastman-Kodak stock solutions of nifedipine and nitren~pine were prepared 90). In addition a PE-50 tubing was inserted into the femoral fresh imm~iately before the experiments, but this did not artery and the cannula tunneled under the back skin, exited enhance the inhibitor action of the dihydropy~dines. The at the nape and further secured by an adhesive collar and w-conotoxin was added from concentrated stock solutions a spring wire as previously reported.” The rats were allowed in water (or in 0.9% NaCl solution for the in uiuo experito recover in their home cage with food and water ad libitum ments), which were prepared immediately before use. Cell for l-3 days. On the day of the experiment the arterial stimulation with high K+ was carried out by adding KC1 catheter was connected to a pressure transducer (RP 15OOi from a 3.5 M stock in Solution A (or in Solution C for Narco) for arterial pressure (mean) and heart-rate recording experiments carried out in Na+ -free medium). Hyperfor 3Wmin to document the basal levels of these variosmotic additions of NaCl, at the concentrations used for ables. Thereafter, o-conotoxin was injected in a lo-p1 high K+ stimulation, did not affect the fluorescence signal volume through the guide cannula by a pre-measured of FURA-2-loaded cells. In the experiments with EGTA (7.5 mm) 27-g needle. Systematic hemodynamic and behavillustrated in Fig. 8, EGTA was added to Solution A from ioral responses were monitored continuously for 2-3 h. a stock solution containing 75mM EGTA and 0.25M Blood samples (200~1) were withdrawn from the arterial Na-HEPES (pH 7.4). line just prior to 120 min after w-conotoxin administ~tion for blood pH, ~0, and pC0, analysis (Corning, PA). RESULTS

Cell depolarization with high K+ (25 mM) gave rise to a very rapid increase in FURA- fluorescence, followed by a much slower decrease towards a “plateau” (Fig, la). Shown underneath the fluorescence

The fits of some of the data (e.g. in Figs 2 and 5) to

the Boltzmann distribution function [see eqn (3) below] were carried out by non-linear regression analysis based on the Marquardt algorithm. Smoothing of some fluorescence Det

EGTA

1

I

25K*

ACh IWuMt

7 ti

a

b

25K’

Fig. 1. ChromafXn cell intracellular Ca2+ responses to high K+ and acetylcholine. Fluorescence of FLJRA-Zloaded chromafIin cells was monitored with excitation and emission wavelengths set at 340 and 510 nm, respectively, as explained in Experimental Procedures. Left (a, c) and right records (b, d) represent measurements carried out using two different samples from the same batch of FURA-2-loaded cells. Cells were stimulated with 25 mM KC1 (25 K+) and 80 FM acetylcholine (ACh) as indicated by the arrows. To calibrate the fluorescence signals in terms of [Ca’+](, the non-ionic detergent C,,E, (1 mM; Det) was added to disrupt the cells, followed by EGTA (4 mM) (see Experimental Procedures). Shown underneath the fluorescence records are the respective plots of [Ca2+], versus time, generated off-line using a microprocessor. [Ca’+], values were calculated using eqn (1) (see text).

record is the respective microprocessor-generated plot of [Ca’+], versus time (Fig. Ic). It may be seen that depolarization caused [Ca’+], to increase from a resting level of c. 120 nM (average 115 + 26 nM; n = 37) to a peak level of c. 290nM. Subsequently towards a “plateau” in an [Ca2+ II relaxed exponential-like fashion (half-time, 80s; average 142 f 60 s, n = 7). This transient character of the chromaffin cell [Ca’+], response to depolarization is in contrast to most previously reported data using Quin-2.8,39,40These previous studies indicated that the elevation of [Ca’+], following depolarization was sustained. Also shown in Fig. 1 (right traces) are the fluorescence and [Ca*+], responses to the natural

A 65K’

cholinergic agonist acetylcholine (ACh). as recorded from the same batch of FURA-2-loaded cells. [C’a-‘- 1, rose rapidly to a peak (c. 140 nM) upon stimulation with ACh, after which it declined to a near-rcstinp [Ca* ‘1, level (half-time, 44 s; average 73 _i_IX s. n = 3). One caution in interpreting these results is that, in some other cells, the hydrolysis products of Quin-2jAM and FURA-2/AM have also been found in non-cytosolic acidic compartments.” This could have been a problem for chromaffin cells. However. the results of Fig. 1 show this possibility to be unlikely under the experimental conditions of this study. For example, if residual FURAwere indeed co-stored with catecholamines in the secretory granule, as a result of local hydrolysis of FURA-2/AM by esterases, any ACh-induced release of probe into the medium would have been detected as a “plateau” of at least the same size as that obtained with high K + This clearly was not the case. Voltage-dependence to high K’

, -20

I

I

qf the intracellular Ca’ + response

To study the modulation by membrane potential of the [Ca’+], response to K+, cell aliquots from the same batch of FURA-2-loaded chromaffin cells were challenged with various K+ concentrations over the concentration range l&65 mM. The size of the initial increase in [Ca’+], brought about by high K + was concentration-dependent (Fig. 2A). It was also apparent that the effect could be recorded at added KC1 concentrations as low as 10 mM. In all instances, the initial increase in [Ca’+], was followed by a relatively slow relaxation towards a “plateau”. Assuming that the initial increase in [Ca*+], reflects solely Ca2+ entry across the plasma membrane and furthermore, no significant intracellular that, buffering and/or efflux of Ca2+ takes place immediately after K+ stimulation (i.e. from beginning of stimulation to peak), the magnitude of the rapid [Ca2+ 1, change (A[Ca’+ I,) can be related to the charge (Aq) carried by Ca’+ ions over the same short period of time as follows:

0

MEMBRANE POTENTIAL, mV

Fig. 2. Voltagedependence of the intracellular Ca’+ response to high K+. In panel A, different cell samples from the same batch of FUBA-2-loaded cells were stimulated with increasing amounts of KC1 (10, 25, 35, 50 and 65 mM) as indicated by the arrows. Time between addition of cells to the cuvette and KC1 addition was 5 min in all cases. The plots of [CaZ+], versus time were generated off-line as indicated in the legend to Fig. 1 and in the Experimental Procedures. In panel B, the magnitude of the initial increase in [Ca*+], induced by high K+ (AICaz+li) is plotted against chromafhn cell membrane potential. Membrane potential was calculated using the Goldman-Hodgkin-Katz equation (see text). [Na+li, w+], and the permeability ratio PNn/PK were taken as 30 mM, 106 mM and 0.07, respectively. The solid line represents the best fit of the data to the Boltsmann distribution function [qn (3)]. The transition potential (V,), effective grating charge (z) and parameter A obtained from the fit were - 23.4 mV, 5.3 and 427.6, respectively. Data are presented as mean f S.D. (n = 3-5).

Aq = 2eN, V,A[Ca’+],.

(2)

where e, NA and V, stand for the electron charge. Avogadro’s number and average cell volume. respectively. Thus A[Ca2+], would be expected to bear a proportional relationship with the amount of positive charge generated by Ca*+ ion translocation within the first few seconds after stimulation. For this reason, A[Ca*+], has been chosen as a suitable parameter to describe the voltage-dependence of the rapid [Ca*+], response to high K’. Accordingly we attempted to plot A[Ca2+ 1, against calculated chromaffin cell membrane potential in Fig. 2B. Calculation of membrane potential for each KC1 concentration was carried out using the modified Goldman-Hodgkin-Katz zero-field equation,‘7.‘9 assuming that Na+ and K+ permeabilities (P,, and P,, respectively) were the only determinants of the

Dihydropyridine-insensitive calcium entry into chromaffin cells diffusion potential created across the membrane.23 For these calculations, ma+],, [K’], and the permeability ratio P,,/PK were taken as 30mM, 106mM and 0.07, respectively.23 A[Ca2+li was found to bear a sigmoidal-like relationship with the calculated membrane potential (Fig. 2B), as expected for a process closely related to activation of voltagedependent channels. Furthermore, A[Ca2+ Ii could be fitted well to the Boltzmann distribution function for two state transitions: Z=A{l

+exp[-zF(V-

V,)/RT]l-’

739

A 8oo\

[Caz*],,mM:

(3)

where, in ion channel terminology, F, R and T have their usual meanings, V is membrane potential, V, (transition potential) is the voltage at which calcium channels are open half of the time, z is the effective gating charge and A is a scaling factor. The transition potential and the effective gating charge calculated from the fit were - 23.4 mV (equivalent to [K+], = 33 mM) and 5.3, respectively. Interestingly, the calculated transition potential is very close to the membrane potential calculated from the external K+ concentration required to elicit half-maximal catecholamine secretion (c. 32 mM43). Extracellular calcium-dependence Ca2+ response to high K+

of the intracellular

In order to study the extracellular Ca2+dependence of the [Ca2+], response to depolarization, cell aliquots from the same batch of FURA-Zloaded cells were added to “Ca2+-free” solution (Solution B) supplemented with various amounts of CaCl, to give final calcium concentrations in the range 0.1-2 mM. Five minutes later, the cells were challenged with high K+ (50 mM). The extent of the initial rise in [Ca2+li, brought about by depolarization, increased with extracellular calcium concentration ([Ca2+],) in a concentration-dependent manner (Fig. 3A). It was also apparent that the transient character of the [Ca2+], signal could only be observed at extracellular calcium concentrations of 0.5 mM or higher. Furthermore, A[Ca2+li did not become saturated in the concentration range 0.1-2 mM (Fig. 3B), in agreement with the the [Ca2+],-dependence pattern of catecholamine secretion.2’,43 The [Ca2+li response observed in the presence of external Ca2+ was totally blocked when EGTA (5 mM) was added to cells suspended in Ca’+containing solution (Solution A), thus reducing Ca2+ concentration to c. 0.17 PM (Fig. 8b). Identical results were obtained when cell aliquots in regular

Ca2+-containing medium were added to an EGTAcontaining solution (Solution C), thus reducing free Ca2+ concentration to sub-nanomolar levels. Cells were also incubated with EGTA (2 mM) for 5 min, challenged with high K+ (50 mM), and finally supplemented with excess calcium to bring [Ca2+],up to c. 6mM. A robust [Ca2’], increase was then recorded (data not shown). Thus, the lack of effect of high K+ on [Ca2+li observed in the presence of EGTA is

[CL++],,. mM Fig. 3. Extracellular Ca 2f dependence of the intracellular Cal+ response to high K+. In panel A, cell aliquots from the same batch of FURA-2-loaded cells were added to Ca2+free solution supplemented with various amounts of CaCl, (0.1,0.2,0.5, 1 and 2 mM). Five minutes later, the cells were stimulated with 50mM KC1 (50K+) as indicated by the arrows. The plots of [Ca2+], versus time were generated off-line as indicated in the legend to Fig. 1 and in the Experimental Procedures. This experiment is representative of two other experiments. In panel B, the magnitude of the initial increase in [Ca2+], induced by high K+ (A[Ca2+],) is plotted against extracellular Ca2+ concentration, [Ca*+],.

unlikely to be due to any deleterious effect of the calcium chelator on cell integrity and function. In addition, these experiments suggest that Na+ entry across voltage-dependent Na channels plays no role either in the earlier or in the later stages of the [Ca2+], response to depolarization. This is because Na channels should be activated by depolarization regardless of the extracellular Ca+ concentration. Modulation of the intracellular Ca2+ response to high K+ by dihydropyridine calcium channel agonist and antagonists

We have used a dihydropyridine (DHP) calcium channel agonist and several DHP antagonists as pharmacological tools to examine the role of DHPsensitive calcium channels in the [Ca2+], changes brought about by depolarization. Addition of (+)202-791, a potent DHP agonist,20~28~36 shortly after stimulating the cells with high KC (25 mM) induced a rapid increase in [Ca2+],

74.0

L. M. ROSARIOYI ~61

400

25K’

25K’

1 300

2bo 3

100 :

a

__&

zi

b 25K’

400

t

G 300

100

25K’

+I-1202-791 (1 +iM)

+kkGO2-791 (1 pM1

c

d

Fig. 4, Detection of dihydropy~di~e receptors in chromatlin cells. Different ceh samples from the same batch of FURA-2-loaded cells were stimulated with 25 mM KCI (25 K+) as indicated by the first arrow of each record. Time between addition of cells to the cuvette and KC1 addition was 5 min in all cases. In trace b, the dihydropyr~dine agonist (+)202-791 and antagonist (-)202-791 were added sequentiaby after KC1 as indicated. In trace c, &is were preincubated with (-)202-791 for 5 min; (+)202-791 was added in the presence of KC1 as indicated. In trace d, cells were preincubated with ($)202-791 for 5 mm; (-)202-791 was added in the presence of KC1 as indicated. The plots offCa2+li versus time were generated off-line as indicated in the legend to Fig. 1 and in the Experimental Procedures. These experiments are representative of 2-3 other experiments.

(Fig. 4b). A slower decay ensued, but the [Ca** Ii levels recorded in the continued presence of the cafcium agonist remained higher than in control (Fig. 4a) over severai minutes. Addition of the DHP calcium channel blocker ( - 1202.791, in the presence of its stereoisomer ( + )202-79 I, rapidly antagonized the agonistic effect (Fig. 4b). ~-incn~ting the cells with the calcium agonist potentiated the early [Ca2+ Ii response to depolarization (Fig. 4d), as expected. However, pre-incubating the cells with the calcium antagonist failed to block the high K+ -induced rise in [Ca2+li (Fig. 4~); instead, a relatively modest 31% reduction of A[&az+& was obtained. The response to the calcium agonist was strongly attenuated when this was added to the depolarized cells in the presence of the calcium an~~nist (Fig. 4~). Two important features of the effects of the catcium agonist and antagonist (-+> and (-)202-791 deserve mention here. First, the increase in [Ca”+Ii brought about by the calcium agonist confirms the existence of DHP receptors in the chromaBn ~eI1.‘~ Second, a high antagonist con~ntrat~on only partially inhi~~d the [Cat+ fi response to de~ta~~t~n. These observations therefore led us to examine in more detail the inhibitory e&&s of DHP calcium ehannel blockers on the [Ca*‘& responses to depolarization. To this end, experiments have been tied out in which cells were pre-incubated with ~fe~pine and nit~ndipine at various co~tra~ons and incu-

bation times. The effects of these blockers on [CaZ”]i signals induced by deplaning steps of d#erent sizes were also studied. Pre-incubating the cells with ~it~ndipi~e (l pm) for 5 mm affected only partially the size of the initial fCa2+li changes induced by either 25 or 65mM KC] (Fig. SA). Thus, in this experiment, ni~en~p~ne reduced A[Ca2+li by c. 37 and 18% at 25 and 65 mM KCl, respectively. For a given size of the defiling stimulus, the extents of the in~bito~ effect of either nitr~ndipine or nifedipine were found to be ~o~~ntrationindependent in the ranges l-5 PM, In addition, they were not found to be de~ndent on the in~ub~~on time, at these ~n~ntrations, in the range 3-30 min Fu~he~ore, when tested at the same incubation time in the co~tration range l-5 PM, both DHP blockers behaved identically (see Pig. 5B). The reIatively small inhibitory effects of ~tr~~~~ and ~fed~~~e were not the result of photodegradation of the DHP molecules during irradiation, as monito~~g the truorescerne by exciting inte~ittentIy for only brief periods, tith the help of a shutter device, did not increase the incitory efficacy of the drugs. Nor were they due to Ga2+ ions entering chroma~ celfs through vol~d~~~~~ Na channels, as suppressing this possible ~&XXX pathway by either p~-t~ti~g the cells with ~~~6~~~ (20 FM) or replacing external Na+ for ~-~thyl~

Dihydropyridine-insensitive

calcium entry into chromaffin cells

741

[Ca2+li response to high K+, thus ruling out involvement of the ACh receptor channel (data not shown). It could also be argued that the blockage efficiency of nitrendipine (or nifedipine) might depend on membrane potential, with the DHP molecule becoming a more effective blocker at more positive membrane potentials.3.24 We have therefore performed the following control experiments. First, KC1 (50 mM) was added to cells in the presence of EGTA (2 mM); next, nitrendipine (1 PM) was added and, 4min later, the cells were challenged with excess calcium (10 mM CaCI,). A robust increase in [Ca2+li ensued (data not shown), indicating that the incomplete blockade of the [Ca2+li response to depolarization reported here cannot be simply explained by the relatively hyperpolarized state of the cells at the time of addition of the DHP blockers. This conclusion could be further supported by examining the effect of nitrendipine (1 p M) on elevated [Ca*+ Ii, 1 min after cells had been challenged with high K+ (50mM). Nitrendipine induced, indeed, a rapid decrease in [Ca2+li, but only to levels comparable to those obtained with cells pre-treated with the DHP blocker (data not shown). Depolarizing stimuli of different sizes may conceivably activate varying proportions of at least two different types of voltage-dependent Ca channels, including the DHP-sensitive channel. For this reason, MEMBRANE POTENTIAL, mV we have compared the [Ca’+], responses obtained at Fig. 5. Modulation of the intracellular Ca’+ response to various KC concentrations, in the range 10-65 mM, high K+ by dihydropyridine Ca channel blockers. In panel to those attained with cells which had been preA, different cell samples from the same batch of FURA-2loaded cells were stimulated either with 25 mM KC1 (25 K+ ; incubated with nitrendipine or nifedipine. Thus cell upper traces) or with 65 mM KC1 (65 K+ ; lower traces) as aliquots from the same batch of FURA-2-loaded indicated by the arrows, both in the presence (right traces) cells were sequentially challenged with increasing K+ or in the absence (left traces) of nitrendipine. Preincubation concentrations, and this sequence was repeated with time with nitrendipine was 5 min in both cases. The plots of cells pre-incubated with either nitrendipine (1 p M) or [Ca2+], versus time were generated off-line as indicated in the legend to Fig. 1 and in the Experimental Procedures. In nifedipine (5 PM). The values of A[Ca2+li for each panel B, the magnitude of the initial increase in [Ca2+], individual experiment are plotted in Fig. 5B against induced by high K+ (A[Ca2+li) is plotted against chromaffin the calculated chromaffin cell membrane potential. cell membrane potential for experiments such as those A[Ca2+li was found to bear a sigmoidal-like reillustrated in A. Circles represent measurements carried out lationship with the calculated membrane potential with cell samples from the same batch of FURA-t-loaded both in control (open symbols) and in the test runs cells, either in the presence (closed circles) or in the absence (open circles) of 1 PM nitrendipine. Squares represent (closed symbols). Fitting A[Ca2+li values obtained measurements carried out with -cell samples from the from the control experiments to the Boltzmann distrisame batch of FURA-2-loaded cells. either in the nresence bution function [eqn (3)] yielded a value of -22.9 mV (closed squares) or in the absence (open squares) bf 5 PM for the transition potential (I’,,) and of 7.1 for the nifedipine. Preincubation time with either nifedipine or nitrendipine was 5 min in all cases. The upper solid line effective gating charge (z). Fitting A[Ca2+], values represents the best fit of control data (empty circles and obtained from the test runs (pre-incubation with squares) to the Boltzmann distribution function [eqn (3)]. nitrendipine and nifedipine) to the same function The transition potential (V,), effective grating charge (z) and parameter A obtained from the fit were -22.9 mV, 7.1 gave a V, value of -22.3 mV and a z value of 10.7. Therefore, dihydropyridine blockers had little effect and 428.8, respectively. The lower solid line represents the best fit of nifedipine and nitrendipine data (closed circles on the voltage-dependence of activation of Ca2+ and squares) to the Boltzmann function. The values of V,, entry into the cytosol of chromaffin cells. It is also z and A obtained from the fit were -22.3 mV, 10.7 and apparent from Fig. 5B that both DHP-like calcium 307.8, respectively. antagonists depressed the activation curve at all membrane potential values tested. However, the o-glucaminate (see Experimental Procedures) failed extent of the inhibition, as calculated from the to enhance the inhibitory efficacy of the blockers fitted curves, was about twice as large for cells (data not shown). Pretreatment of cells with d- stimulated with 25 mM K+ (E = -25 mV; % tubocurarine (10 PM) failed to affect the size of the inhibition = 48%) than for cells stimulated with

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35-65 mM K+ (E = -20 to -7mV; % inhibition = 23-26%). From other experiments, nitrendepine and nifedipine inhibited A[Ca’+& by 51 + 18% (n = 3) and 23 f 14% (n = S), at 25 and 50mM KCl, respectively. Modulation of the intracellular Ca2+ response to high K+ by w-conotoxin GVIA o-conotoxin GVIA is known to block L- and N-type Ca2+ currents in neurons.31-33,37We therefore used this toxin as a probe for the N-type Ca channel in the chromaffin cells. As shown in Fig. 6 the effect of o-conotoxin on the size of the [C!a*+1,response to high K+ (50mM) was small. Pre-incubation of chromaffin cells with 0.1 and 1 PM o-conotoxin for 5 min inhibited the initial increase in [Ca2+li by only 3 and 16%, respectively. In other experiments, the DHP-insensitive component of the [Ca*+ li signal remained unaffected in the presence of w-conotoxin. Thus, when cells from the same batch were simultaneously pre-incubated with o -conotoxin (1 p M) and nitrendipine (I PM), the combined inhibitory effect could not be distinguished from that obtained with nit~ndipine (1 PM) alone. The extent of inhibition of the [Ca2+], signal by w-conotoxin was found to be concentration-independent in the range I-20 p M at pre-incubation times up to 10 min. Evidence for phar~aeo~ogi~ actions of w-conotoxin In order to verify that the small effect of wconotoxin on chromaffin cells was in fact due to shortage of sensitive Ca channels, we tested the toxin on a central rat preparation. In addition to verifying the toxin activity, a complete in vivo analysis of the toxin effect was performed. Injection of o-conotoxin (100 nmol/kg, i.c.v.) to the conscious rat produced a complex behavioral pattern. As early as 2 min after the injection twitching of facial muscles occurred and whisker movements, primarily in the vertical dimension, were followed by shaking of the neck, back, limbs and tail. The shaking

oi_

%ij?

gradually developed into a severe motor incapacitation l&l5 min after o-conotoxin administration. At this time and throughout the experiment (up to 3 h) the rats lost coordinated motor activity and remained in the prone position. A stiff erect tail also marked the typical response. The vigor of the behavior somewhat subsided during the second and third hour, at a time where cyanosis could clearly be seen in the skin and mucous membrane. No fecal excrements were observed and no salivary gland excretions were noticed. The shaking behavior was observed until the end of the experiment. It is noteworthy that this syndrome is reminiscent of the convulsive behavior described in mice injected ICV with purified conotoxin GVIA” or the shaking behavior elicited by o-conotoxins (i.c.v.) in mice.14 Following the w-conotoxin administration, and in parallel to the behavioral response, prominent changes in heart rate, blood pressure and respiratory variables were noticed. An early pressor and tachycardiac response developed over the first 30 min after ru-conotoxin administration, up to 124 & 10 mmHg as compared to 89 + 2 mmHg at the control period (P < 0.05) (Fig. 7). Heart rate increased to 549 Itc 16 beats/min as compared to 407 + 24 beatsjmin at the control period (P < 0.05). However, the increase in blood pressure subsided at 60-90 min and hypotension gradually developed (Fig. 7). In each rat the hypotension resulted in severe shock and ultimately death. Heart rate, however, continued to be extremely high throughout the experimental period. Analysis of blood samples withdrawn 30min after w-conotoxin administration (at the time of peak shaking behavior) revealed no change in pH or blood gases. However, 120 min after w-conotoxin administration, severe hypoxemia developed: p02 fell to 75.6 + 4.3 mmHg (compared to 98.7 rfi 13.6 mmHg in the control period, P < 0.05, n = 3). At this time, no consistent changes were observed in pCOz or pH. Thus, cardiovascular and respiratory collapse marked the terminal stages of the brain intoxication by o-conotoxin.

:o-CONOTOXIN

(0.1 pMI

+ad.ZONOTOXIN (1 PM)

Fig. 6. Modulation of the intracellular Ca*+ response to high K+ by w-conotoxin GVIA. Different cell sampIes from the same batch of FURA-Zloaded cells were stimulated with 50 mM KC1 (50 K*) as indicated by the arrows, either in the presence (two right records) or in the absence (left record) of o-conotoxin. Preincubation time with 0.1 and 1 pM o-coaotoxin was 5 min in both cases. The plots of [Caz+Ii versus time were generated off-line as indicated in the legend to Fig. 1 and in the Ex@mental Procedures. This experiment was representative of two other experiments.

Dihydropyridine-insensitive

143

calcium entry into chromaffin cells

Y,v’#& 672 29 3ii

I

1

CONTROL 0

I

I

I

1

1

I

I

5

15

30

45

60

75

90

I

120

TIME (min) Fig. 7. Effect of oconotoxin administration (i.c.v.) on blood pressure and heart rate of the conscious rat. The recording provides a typical example of the cardiovascular responses to o-conotoxin (100 nmol/kg) injected i.c.v. MAP = mean arterial pressure; HR = heart rate. Time 0 denotes time of toxin injection. Peak pressor response was seen 15min after oconotoxin injection and shock ensued throughout the

second hour.

Modulation of the intracellular Ca2+ response to high K+ by inorganic Ca channel antagonists

We have also used different inorganic Ca channel antagonists to probe the processes involved in the depolarization-induced [Ca2+ Ii signals in chromaffin cells. Among the different divalent cations tested (co2+, Cd2+ and Mg2f), only Mg2+ proved to be amenable for study, as both Co’+ and Cd’+ strongly interfered with the fluorescence assay (see Experimental Procedures). However, the Mg2+ effect could not be readily quantified, as the cation was found to interfere with the fluorescence calibration procedure (see Experimental Procedures). Nonetheless, and somewhat surprisingly, preincubation of cells with MgCl, (20 mM) had only a slight inhibitory effect, if any, on the initial fluorescence increase induced by 50mM K+ (data not shown). On the other hand, M&+ (20mM) is known to inhibit high K+-stimulated 45Ca2f uptake and catecholamine secretion by 70 and 60%, respectively.21 We have also tested two trivalent cations of the lanthanide series, La’+ and Gd3+. La3+ (2-40pM) induced a slow, but steady, concentration-dependent rise in fluorescence (data not shown). In the light of these data and of the ill-defined secretagogue-like effects of La3+,’ we have explored instead the properties of Gd3+ as a potent calcium antagonist.6 Gd3+ slightly decreased the fluorescence (Fig. 8d), an effect reminiscent of the fluorescence quenching observed with FURAsolutions (see Experimental Procedures). The rapid [Ca2+li response seen in the control experiments (Fig. 8e, f) was totally suppressed when K+ was added in the presence of Gd3+, but a slow fluorescence increase ensued (Fig. 8d). Gd3+ was also tested after cell stimulation with

high K+. Thus, addition of Gd3+ 20s after K+ stimulation resulted in a rapid fluorescence decrease, which proceeded to a level about half-way between resting and peak fluorescence values (Fig. 8e). Fluorescence then decayed slowly to control values along a time course of several minutes (Fig. 8d). When Gd3+ was added at a later time point after K+ stimulation (300 s), an identically rapid initial decrease in fluorescence occurred (Fig. 8f). The fluorescence level reached immediately after Gd3+ addition was now closer to resting levels than when Gd3+ was added at the earlier time point. Furthermore, the slower phase of fluorescence decay observed in Fig. 8e was not present. Deconvohition of the fluorescence-time data from the sloti upward drift observed in control (Fig. 8d) revealed, however, that the net effect of Gd’+ was to reduce [Ca2+li towards control values along a slow time course of minutes. From a larger pool of experiments, the average time course of this slow process was estimated by deconvoluting likewise the fluorescencetime data in the terms runs from the fluorescence changes observed in control runs. Lag times between Gd3+ (40 p M) and K+ (50 mM) additions were made to vary in the range 20-300 s in steps of 30, 50 and 100 s. No correlation was found between the halftime of the slow fluorescence decay and these lag times (data not shown). An average half-life of 208 f 81 s (n = 7) was calculated. The information obtained with Gd3+ was confirmed by reducing Ca2+ influx with EGTA in the continued presence of high K+ (Fig. 8~). Thus, addition of EGTA, which in control (Fig. 8b) totally suppressed the [Ca2+li response to high K+, reduced fluorescence rapidly to values about half-way between resting and peak levels. Similarly to the experi-

744

L. M. R~SARIO et ui.

Fig. 8. Modulation of the intracellular Ca2+ response to high K’ by EGTA and Gd’+. Fluorescence traces a, b and c represent experiments carried out on different cell samples from the same batch of FURA-2-loaded cells. In trace b, the response to stimulation with 50mM KC1 @OK+) seen in -@control (trace a) was abolished uoon addition of 5mM EGTA. In trace c, EGTA (5mM) was added in the presence of KC1 as indica&d. In traces b and c, addition of EGTA from a concentrated stock solution (see Experimental Procedures) caused an additional slight decrease in fluorescence due to dilution of the fluorophore. To correct the records for this a&factual change, the fluoresceme data subsequent to EGTA addition were multiplied by an appropriate factor (1.07) point-by-point. Fluorescence traces d, e and f represent experiments carried out on different cell samples from a distinct batch of FURA-24oaded cells. In trace d, cells were s~muFat~ with 50 mM KC1 in the presence of GdCl, . In traces e and f, GdCI, was added to cells 20 and 300 s after st~m~ation with KCl, respectively. These experiments were representative of 34 other experiments.

ments with Gd3+, fluorescence then fell slowly to control levels along a slow time course of minutes. DlSCUSSlON

This study indicates that the initial rise in [CaZ*]i brought about by depolarization is mainly, if not

entirely, due to activation of volta~~~ndent Ca channels. Our results show that de~la~~~on with high K+ elicits a very rapid rise in [Ca2+li, which is followed by a much slower decay towards a “plateau”. We also found that the size of the initial increase in [Ca*+], varied with calculated membrane potential in a sigmoidal-like fashion, and could be well fitted to a Boltzmann distribution function with a transition potential of c. -23 mV. Finally, we learned that the K+-induced [Ca2+J signal could only be observed in the presence of extracellular Ca*+, and it varied with fCa2+f0 in a dose-dependent fashion. This signal was effectively blocked by low concentrations of the lanthanide ion gad~in~um, which has also been reported to act as a potent blocker of both %a*+ uptake by, and cateeholamine secretion from, chromaffin cell~.~ Our data also emphasize the transient character of the chromaffin cell [Ca2+li response to depolarization. In agreement with one reporLz7 but contrary to others in which depolarization with high K+ was reported to elevate [Ca2+li sustainedly,8’39@’our re-

sults show that, following an initial rise, [CaZ*li then decays slowly but signiflcantiy to a “plateau”. Thus, our results would agree better with recent studies showing that a peak increase in 45Ca2+ uptake is followed by inactivation of the uptake, and thus by inference inactivation of voltage-dependent Ca channels.’ However, these later studies aIso indieate that Ca2+ uptake inactivates ~~~nti~y within the first 15-25s after cell stim~a~on with high K+. Is there an important difference between the slow kinetics of the [Ca2’li decay reported here and the fast kinetics of inactivation of 4sCa2+ influx reported by others? We think not for the following reason. The studies with 45Ca2f may report solely on influx from external sites, while our FURA- studies report on at least the sum of Ca* + efflux and infiux from both external and internal sites. Therefore, direct comparison is not warranted. Indeed, it is very likely that the stow decay in cCa*+], could in fact regect simultaneous extrusion of internal Ca*+, superimposed on sustained Ca*’ influx from extraceIlular and int~~llu~r sites. If influx from the medium were occurring we might expect addition of either EGTA or Gd3+ in the presence of high K+ to affect intracellular Ca2+ levels appreciably. This was clearly observed (Fig. 8e, f1. It is also possible that depolarization4nduo Ca2* entry might set off a secondary series of reactions leading to Ca*+ release from internal stores. Such a process could explain the fact that addition of EGTA

Dihydropyridine-insensitive

calcium entry into chromaffin cells

cells resulted in only and Gd’+ to Kc-stimulated incomplete suppression of the [Ca2+ ]i transient. Interestingly, recent reports indicate that high K+ stimulation induces a rapid breakdown of phosphoinositide lipids in pancreatic /?-cells and bovine chromaffin cells, with the concomitant intracellular This metabolite could, accumulation of 1,4,5-IP,. 4,13,4’ in turn, induce Ca2* release from an intracellular Ca2+ ~001~~such as the endoplasmic reticulum or the recently proposed “calciosome”,46 thus accounting for our results. In further support of this hypothesis, we have recently observed that preincubation with caffeine (20 mM) accelerates the decay of the K+-induced [Ca2+li transients. It is as if caffeine were impairing Ca2+ release from intracellular stores, thus limiting the extent of the presumed Ca2+ amplification process (L. M. Rosario and H. B. Pollard, unpublished observations). In addition, our data may help to establish the relative roles of different routes for Ca2+ entry across the depolarized chromaffin cell membrane. Indeed, we have confirmed that chromaffin cells are equipped with dihydropyridine (DHP) receptors.i6 Yet, several calcium antagonists of the DHP family [( -)202-791, nifedipine, nitrendipine] had only a relatively moderate effect on the [Ca2+li response to depolarization. This cannot be explained by the magnitude of the nifedipine and nitrendipine concentrations used here, which were 30- to 1400-fold higher than the reported cater&l values for inhibition of K+-stimulated cholamine release.15 ([ ‘H]Nitrendipine has been shown to bind to membrane fragments from bovine adrenal medulla with an apparent Kd of 1.2 nM.i6) A possible alternative explanation could be that, as in other systems, DHP antagonists appear to be more effective blockers of Ca channels when added to depolarized rather than to resting cells.3,24We rule out this possibility on several grounds. First, a robust [Ca2+li increase could still be observed with cells that were preincubated with nitrendipine in the presence of high K + and in the absence of external Ca2 +, and then challenged with excess calcium. Second, addition of DHP blockers after K+ stimulation failed likewise to reduce [Ca2+], to resting levels. Last, a relatively constant extent of inhibition (c. 25%) was observed over a wide range of membrane potential values (- 20 to - 7 mV). Therefore, the results suggest that, in addition to the DHP-sensitive, voltage-dependent Ca channel, chromaffin cells are provided with at least another voltage-dependent Ca2+ entry pathway. Different types of voltage-dependent Ca channels have recently been described using voltage-clamp methods.32.33The DHP-sensitive L-type current is a slowly inactivating Ca2+ current with a high voltage threshold for activation. In neurons, muscle and other tissues, two other types of Ca2+ currents have been found, i.e. the Nand T-currents.“-“s’* The later is a rapidly inactivating and DHP-insensitive Ca2+ current with a lower voltage threshold for activation than the

745

L-current. In neurons, the Conus geogruphus toxin o-conotoxin”“‘34,35 has been shown to block potently the N- and L-type Ca2+ currents, but not the T-type current.3’~33~37 We have therefore had recourse to this toxin in an attempt to find an N-type Ca channel in the chromaffin cell. However, w-conotoxin had only a small inhibitory effect on the [Ca2+li transients elicited by depolarization. Furthermore, w-conotoxin failed to inhibit the DHP-insensitive component of the [Ca2+], transient, indicating that the chromaffin cell lacks an o-conotoxin-sensitive, neuronal-type Ca channel. The vivid activity of this toxin when administered to the central nervous system of the rat in vivo indicates that the N-type channels found in brain are not present in chromaffin cells. The possibility cannot be ruled out, however, that the chromaffin cell might also be equipped with an as yet uncharacterized T-type Ca channel. Our finding that most of the [Ca2+li signal induced by depolarization remained unaffected by DHP blockers is in apparent disagreement with the observation that some DHP blockers profoundly inhibit K+-induced catecholamine secretion. Thus, nitrendipine (1 PM) has been reported to inhibit high K+-stimulated [ ‘Hlnorepinephrine release by c. 85%.9 A clue to understanding the apparent discrepancy with our results may lie in a recent study by Boarder et ~1.~showing that a DHP blocker of similar potency to nitrendipine [( +)nicardipine] blocked high K+-induced [ ‘Hlepinephrine and [ ‘Hlnorepinephrine less extensively, i.e. by c. 65 and 75%, respectively. Furthermore, the inactive stereoisomer (-)nicardipine was found to have non-specific inhibitory effects at relatively high blocker concentrations (c. 1 PM).’ It is therefore likely that the DHP-insensitive component of the K+ -induced catecholamine secretion may be longer than previously thought, i.e. 30% or more. Yet, in our study, DHP blockers were found to inhibit K+-induced [Ca2+li signals by less than 50%. It can therefore be concluded that the DHP-sensitive Ca channel (L-type channel) may be more relevant for secretion than other channel types. It could be that the L-type channel might be concentrated in “hot spots” strategically located to set off exocytosis by driving Ca2+ preferentially to exocytotic sites. Or, alternatively, the L-type Ca channel in these “hot spots” might be more efficient in triggering “Ca2+-induced Ca2+ release” than other Ca channels. CONCLUSION

We propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of CaZt release from an intracellular store, and that this process may be relevant for catecholamine secretion by the chromaffin cell. We also propose the involvement of at least two different classes of voltage-dependent Ca channels in the chromafbn cell response to depolarization.

746

t. M.

%.XARlU

~c~~o~~e~~e~~r~-The skillful technical assistance of Mrs Diane Seaton with the chromaffin cell preparation is gratefully acknowl~ged. Thanks are also due to Dr A. Stutzin for help with the computer program used to generate the fits. The authors are grateful to Dr E. For&erg

et

ai

for many discussions and suggestions, and for readmp the ma&script. This study was supported in part hi grants from, USAMRDC (G19231) and from U.S. Spain Joint Committee, National Science Foundation No CC%8409-002.

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in brain. Probable components of the calcium channel. J. b&l. Chem. 262, 9877-9882. Artaieio C. R.. Garcia A. G. and Aunis D. f1987) Chromaffin cell calcium channel kinetics measured isotoDicallv . I throu& fast c&urn, strontium, and barium huxei. J. biQf. Chem. 262, 915-926. Bean B. P. (1984) Nitrendipine block of cardiac calcium channels: high&i&y binding to the inactivated state. Proc. n&n. Acad. Ski. U.S.A. 81, 6388-6392. Biden T. J., Peter-Riesch B., Schlegel W. and WolIheim C. B. {1987) Ca2+-mediated gene~t~ou of in&o1 1,4,5-trisphospha~ and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets, Studies with K*, glucose, and carbamyicholine. J. biol. Ckem. 262, 3567-3571. Boarder M. R’., Marriott D. and Adams M. (1987) Stimulus-secretion coupling in cultured chroma%n ceils, Dependency on external sodium and on d~hydrop~djne-sensitive caicium channefs. fliocrirem. Pharmae. 36,163-167. Bourne G. W. and Trifaro J. M. (1982) The gadolinium ion: a potent blocker of calcium channels and catechcrlamine release from cultured chroma%m cells. Neuroscience 7, 1615-1622. Bresnahan S. J., Baugh L. E. and Borowitz J. L. (1980) Mechanisms of La3.+-induced adrenal catecholamine release. Res. Commun. Chem. Pathol. P/tarmac. 28, 229-244. Burgoyne R. D. and Cheek T. R. (198.5) Is the transient nature of the secretary response of chromaffin cells due to inactivation of calcium channels? Fedn Eur. biochem. Sacs Lett. 182, 115-I 18. Cena V., Nicolas G. P., Sanchez-Garcia P.. Kirpekar S. M. and Garcia A. G. (1983) Pharmacological dissection of receptor-associated and voftage-sensitive ionic channels involved in catecholamine release. Neurosciernfe 1% 1455-1462. Clark C., Oiivera B. M. and Crnz L. J. (1981) A toxin from the venom of the marine snail Cunlts geogrupk which acts on the vertebrate central nervous system. Toxicon 19, 591-599. Cruz L. J. and Olivera B. M. (1986) Calcium channel an~gonists. 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(1943) Potential, impedance and rectification in membranes. J. gen. Physkd 27, 37-60. Grynkiewia G., Poenie M. and T&n R. Y. (1985) A new generation of Ca 2* indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440-3450. Hodgkin A. L. and Katz 8. (1949) The effect of sodium ions on the electrical activity of the giant axon of the quid. f. Phy~~o~.108, 3-A-77. Hof R. P., Ruegg U. T., Hof A. and Voget A. (1985) Stereoselectivity at the calcium channel: opposite action of the enantiomers of a 1,4-d~hydropy~di~e. J. cardiooasc. Pharmuc. 7, 689-693. Holz R. W., Senter R. A. and Frye R. A. (I9821 Relationship between Ca*+ uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla. $. ~e~roc~e~. 3% 635646. Hoshi T. and Smith S. J. (1987) Large depolarization induces long openings of voltage-dependent calcium channels in adrenal chroma&in cells. J. Neurosci. 7, 571-580. lshikawa K. and Kanno T. (1978) Influences of extracellular calcium and potassium concentrations on adrenaline release and membrane potential in the perfused adrenal medulla of the rat. Jup. J. Physiol. 2% 275-289. Kamo T. J. and Miller R. J. (1987) Voltage-dependent nitrendipine binding to cardiac sarcolemmal vesicles. M&c. Pharhac. 32, 278-285. Kidokoro Y. (1985) ~iectrophyslolo~ of adrenal chromaflin cells. In The E~e~trophysjo~ogyof the Secretory Cell feds Poisner A. M. and Trifaro J. M.), pp. 195-218. Elsevier Science, Amsterdam. Kirshner N. (1987) Sodium and calcium channels in cultured bovine adrenal medulla cells. in Sfimuius-Secretion Coup&g in Chrorna~~ Ceils (eds Rosenheck K. and Lelkes P. I.), pp. 71-86. CRC Press, FL. Knight D. E. and Kesteven N. T. (1983) Evoked transient intracellular free Ca ** changes and secretion in isolated bovine adrenal medullary cells. Proc. R. Sac. B218, 177-199. Kongsamut S., Kamp T. J., Miller R. J. and ~nguinetti M. C. (1985) Calcium channel agonist and antagonist e%cts of the stereoisomers of the dihydropy~dine 202-791. Bio&em. HQphys. Res. CQmmtm. H%, 141-148. Kuijpers G. A. J., Rosario L. M. and Omberg R. L. Role of intracellular pH in secretion from adrenal medulla chromat%n cells; effects of weak acid and base. J. biol. Chem. (in p&s). Malgaroli A., Milani D., Meldolesi J. and Pozzan T. (1987) FURAmeasurements of cytosolic free Ca”’ in monolayers and suspensions of various types of animal cells. J. Cell Blot. 1W, 2145-2155. McCleskey E. W., Fox A. P.. Feldman D. l-l., Cruz L. J., Olivera B. M., Tsien R. W. and Yoshikami D. (1987) Omega-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle. Prot. natn. Acad. Sci. U.S.A. M. 4327-4331.

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