J. Physiol. (1976), 262, pp. 743-753 With 1 plate and 3 text-ftgure8 Printed in Great Britain
743
ELECTRICAL EXCITABILITY OF CULTURED ADRENAL CHROMAFFIN CELLS
BY BERNARD BIALES, MARC DICHTER AND ARTHUR TISCHLER From the Departments of Neurology and Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.
(Received 10 May 1976) SUMMARY
1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro-electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded - 50 mV. 3. Chromaffin cells were capable of generating all-or-nothing overshooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 106 g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae. INTRODUCTION
The chromaffin cells of the mammalian adrenal medulla are catecholamine secreting cells derived from the neural crest. They receive a cholinergic preganglionic sympathetic innervation, mainly through the splanchnic nerve and are thus homologous with post-ganglionic sympathetic neurones. Acetylcholine (ACh) has been shown to stimulate catecholamine secretion in vivo (Douglas, 1975) and to depolarize chromaffin cells in vitro (Douglas, Kanno & Sampson, 1966, 1967 a, b). Notwithstanding the similarity of chromaffin cells to neurones, previous studies using microelectrode penetrations have found that chromaffin cells have low resting potentials both in vivo and in vitro and do not generate action potentials 26
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B. BIALES AND OTHERS (Douglas et al. 1967 a, b; Kanno & Douglas, 1967; Matthews, 1967; Fawcett, 1969). We have performed similar experiments on gerbil and human chromaffin cells in vitro and on the gerbil tissue slices. Our results demonstrate that chromaffin cells can maintain resting potentials greater than -50 mV and are capable of generating short duration all-or-nothing overshooting action potentials, both in response to direct electrical stimulation and to iontophoretic application of ACh. A preliminary report of these results was given at the 1975 Annual Meeting of the Society for Neuroscience. At the same meeting, Hagiwara & Kidokoro also reported findings of chromaffin cell excitability and ACh responsiveness. 744
METHODS Culture methods. For each dissociation, four to ten adult gerbil adrenal glands were aseptically excised, trimmed of fat, and sliced at the poles to allow access of enzymes. Sequential digestion was carried out under 95% air-5 % CO2 at 360 C in 2 ml. trypsin (Armour Pharmaceutical) and 1-2 ml. collagenase (Sigma) for 45-60 min each (both enzymes at 0.1 % in Eagle's MEM with 20 u. penicillin/ml. and 20 #sg streptomycin/ml., GIBCO). Between the two digestions the adrenal cortex was cut away and any mutilated medullae were discarded. After digestion, the tissue was placed in MEM with antibiotics, 200 mg % glucose and 10% rat serum (filtered through 20 jsm Nalgene filter and heat inactivated at 56° C for 30 min) and triturated successively through normal and flame-constricted Pasteur pipettes. Yield was typically 3000 cells per medulla. The cell suspension was spun down at 220 g for 4 min and resuspended in a small volume of the above medium plus 20-30 ng/ml. nerve growth factor (NGF) (kindly supplied by Dr Lloyd Greene) and 16 ,ug corticosterone/ml. (Sigma). The cells from j-2 medullae were plated in a volume of 0.1 ml. into a stainless-steel ring (4j mm i.d.) standing in a 35 mm plastic tissue culture dish (Falcon) containing 1-8 ml. medium outside the ring. The dishes were either untreated, coated with gelatin or collagen, or with the cells of a primary or secondary culture of rat embryonic lung previously treated with 10-5 M cytosine arabinoside to inhibit cell division. On the day following plating the restrictor ring was removed. Dishes were generally used within 3 days, but when kept beyond that, the medium was changed at 4 to 5day intervals. For experiments utilizing formaldehyde induced fluorescence, adrenal cells were plated on glass cover-slips previously coated with gelatin or lung cells. For human cultures, single adrenal glands were obtained from a 38 yr old man undergoing nephrectomy for hypertensive renal failure, a 57 yr old woman undergoing adrenalectomy for metastatic breast carcinoma (histological examination revealed no metastatic lesions) and a 61 yr old man with Cushing's disease. Dissociation and plating were carried out as described above except that the medullary regions were excised before trypsinization, digestion periods were extended to 75 min each and the inoculum was raised to 9000-27,000 cells. For recording from adrenal slices, adult gerbil adrenal medullae were excised, dividedand wedged, cut side up, in a block of saline-agar. Penetrations were guided
by eye. Electrophysiology. Micro-electrode penetrations were made in physiological saline (Na 147, Cl 153, K 3 4, Ca 1-8, Mg 1.0, H2PO4 0 4, HPO4 2-4, glucose 8 mm, pH 7.3) with phase contrast visualization at 31-35° C. To allow the addition of Co to the
CHROMAFFIN CELL ELECTRICAL EXCITABILITY
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medium, a similar Tris (2-5 or 5-0 mm at pH 7-2) buffered saline without phosphate was used. Micro-electrodes were made from Corning 7740 glass tubing (o.d. 1 mm, i.d. 0-8 mm) using a modified Kopf vertical puller. They were first filled with double distilled water by boiling for 5-12 min, then back-filled with 3-5 N-K acetate, using a 30-gauge needle. Electrodes were allowed to equilibrate for at least 11 hr before use. Best results were obtained with 40-120 MO electrodes. ACh chloride (1 M, Sigma) electrodes were made in the same manner. Electrodes were connected to an electrometer amplifier (WPI M701 or M4A) by a chlorided Ag wire. Current was carried to ground through an agar-saline bridge to a Ag-AgCl electrode in a 3 N-KC1 bath. Output from the amplifier was displayed on a dual beam oscilloscope (Tektronix) and a multichannel chart recorder (Gould 260). Provision was made for simultaneously passing steady currents and square-wave pulses through the recording micro-electrode by way of a circuit built into the WPI pre-amplifier. The cells were stimulated by either a 40-50 msec square-wave of depolarizing current injected into the cell or by a 0-5 msec pulse injected into the cell (Text-fig. 1 D, E). For ACh iontophoresis, ACh-filled micro-electrodes (tip 0-5 lam, R =100-200 MO) were positioned under microscopic control to within 5 #sm of the cell being stimulated. Most electrodes required a hyperpolarizing 'holding' current of between 1 and 5 nA to prevent depolarization as the electrode approached the cell. Iontophoretic responses were sought with short (1-4 msec), 0-2-25 nA depolarizing pulses superimposed on the steady 'holding' current. Current was monitored through the WPI pre-amplifier. (ell identification. The chromaffin reaction was performed on cultures fixed for 10-14 hr, in a solution of K2Cr2O4 and K2CrO4 according to the method of Hillarp & Hokfelt (1955). For formaldehyde induced fluorescence, cover-slip cultures of adrenal medulla cells were rapidly blown dry at room temperature and then further air dried over Pr05 for at least 3 days. They were then heated at 800 C for 3 hr over paraformaldehyde which had been equilibrated at 60 % humidity according to the method of Falck, Hillarp, Thieme & Torp (1962). The cover-slips were examined with an AO fluorescence microscope equipped with a BG12 excitor and 470 barrier filter. Electron micro8copy. Cultures were fixed in 3% glutaraldehyde (TAAB), pH 7 3, in tissue culture medium and post-fixed with 1 % osmium in 0-1 phosphate buffer at pH 7-0. Ultrathin sections (80-100 nm) were stained with uranyl acetate and lead citrate. RESULTS
Cell morphology and identification in culture Living chromaffin cells could be recognized in culture by phase contrast microscopy (PI. 1A, B). Single cells initially appeared as spheres (about 8-30 ,sm diameter), but later often flattened or assumed a spindle shape. They had finely granulated cytoplasm with a medium sized reticulated nucleus and small nucleolus. Cells were often seen in clusters and although groups of cells had a characteristic range of morphology, it was sometimes difficult to pick out the boundary of individual cells in the clusters. The identification of the chromaffin cells in vitro was established by a variety of histological techniques. The cells showed intense green fluorescence following treatment with formaldehyde vapour (P1. 1 C), indicating catecholamine content. The cells also exhibited a positive
746 B. BIALES AND OTHERS chromaffinreaction (P1. ID). Finally, electron microscopy of two cellswhich were penetratedwith micro-electrodes and which exhibited action potentials (see below) demonstrated large (typically 0-2 mcm) dense core granules characteristic of chromaffin cells (Coupland, 1965) (PI. 1E).
Electrophysiology Results of intracellular recordings in both gerbil and human cultures were similar. A few cells had membrane resting potentials of -50 to -70 mV, usually attained after spontaneous improvement from an initially lower potential. These cells exhibited all-or-nothing, overshooting action potentials either spontaneously or in response to membrane depolarization (Text-fig. 1A-E). This most active group of cells A
D
C
c
\
E
F.
''
Text-fig. 1. Chromaffin cell action potentials. A, human cell in culture. B, gerbil cell in culture. C, gerbil adrenal medulla slice with one subthreshold and one suprathreshold depolarizing pulse. D, action potential evoked by short depolarizing pulse. E, action potential as in D but with swept out trace. F, repetitive firing with prolonged depolarizing pulse. Calibration: 10 mV and 5 msec (except 0 5 msec for E). Stimulus currents: A, 0-33 nA; B, 0-14 nA; C, 0-28 nA; F, 1-25 nA.
also showed repetitive firing when depolarized beyond threshold (Textfig. iF). More commonly, the chromaffin cells had resting potentials in the range of -10 to -40 mV and action potentials could be elicited by depolarization subsequent to background hyperpolarization of the cells. In the most active cells, rates of rise of the action potential reached 100-220 V/sec and average spike duration at half-height was 2 msec. The action potentials were followed by a hyperpolarizing after-potential. Gerbil chromaffin cells maintained in the presence of NGF (10 ng/ml.),
CHROMAFFIN CELL ELECTRICAL EXCITABILITY 747 corticostorone (17 mcg/ml.), NGF and corticosterone, or without either additive, all demonstrated similar action potentials. In order to be certain that the ability to generate action potentials was not an artifact induced by maintaining the chromaffin cells in vitro, we recorded from chromaffin cells in slices of freshly isolated adult gerbil adrenals. Penetrations were more difficult than in vitro but we were able to observe numerous chromaffin cells which were capable of generating action potentials (Text-fig. 1 C). We cannot comment on mean resting potentials of the cells from which we recorded for several reasons. The cells were relatively small and fragile and most showed extensive signs of injury after penetration - either morphological or electrophysiological. In addition we used relatively high resistance micro-electrodes (up to 150 MD) with variable, and sometimes high, tip potentials. However, it was clear that at least some cells were capable of sustaining high resting potentials. We cannot tell from our data whether the relatively low values for resting potentials which we often obtained and which others have reported (Douglas et al. 1967a, b; Matthews, 1967; Fawcett, 1969) represent the true situation in sitn or are artifacts due to penetration injury. We suspect the latter. One reason for this suspicion is the fact that so many of our 'poor' cells which initially appeared inexcitable could be induced to fire action potentials by background hyperpolarization. Induction of such action potentials has never been reported as an artifact and does not occur in many other nonexcitable tissues. For similar reasons we have not specified the percentage of chromaffin cells which could generate action potentials. These data are meaningless if they include all the injured cells. Although we cannot rule out the possibility that some cells are inexcitable, in four experiments where we tried to deal with this question, thirty out of thirty sequentially penetrated human cells and fourteen out of fifteen gerbil cells could be induced to fire action potentials. In total we recorded from well over 300 electrically active chromaffin cells. Ionic basis of the action potential The chromaffin cell action potential disappeared in tetrodotoxin (TTX) at 10-7 to 10-6 g/ml. TTX is known to block most Na spike mechanisms (Narahashi, 1974). These experiments were performed by sequentially penetrating different cells in control medium, TTX-medium, and back in control (after four washes). In every case it immediately became impossible for the chromaffin cells to generate action potentials in the presence of TTX. (In a few cells in TTX, 10-7 g/ml., there were small (ca. 10 mV) potentials which occurred on top of the passive depolarizing charging curve. It was difficult to distinguish these from delayed
748 B. BIALES AND OTHERS rectification. These small extra potentials were not present in TTX, 10-6 g/ml.) After washing the chromaffin cells with control medium the cells were again regularly able to generate action potentials. Text-fig. 2A is an example of a human chromaffin cell with a resting potential of -30 mV and an input resistance of approximately 90 MQ which did not show an active response in TTX (t0-7 g/ml.) despite background hyperpolarization. Text-fig. 2 B shows a re-penetration of the same A
B
c
D
Text-fig. 2. Ionic basis of chromaffin cell action potential. A, large depolarizing pulses produce no active response in presence of tetrodotoxin (10-6 g/ml.). B, same cell as in A after removing TTX. Resting membrane potential and input resistance were lower but cell could still generate action potentials. C, repetitive spikes from chromaffin cell in Ca-free medium. Upper trace indictes zero base line and duration of intracellular current pulse. D, chromaffin cell action potential in presence of CoCl2 (10 mm). Calibration: 50 mV and 25 msec for all traces. Stimulus currents: A, 1-8, 2-5 nA; B, 1-1 nA; C, 0-21 nA; D, 0-24 nA.
cell in control which then showed somewhat inferior resting membrane parameters (low resting potential and input resistance approximately 50 MQ) but which could generate all-or-nothing action potentials. The above results indicate that the chromaffin cell action potentials are most likely generated by a Na mechanism. Since Ca ions have been shown to be necessary for adrenal medullary secretion (Douglas, 1975), we attempted to determine if Ca ions were necessary for action potential generation. In medium to which no Ca had been added, the chromaffin cells were still capable of generating large action potentials. (Although we will refer to this as 'Ca-free' medium,
CHROMAFFIN CELL ELECTRICAL EXCITABILITY 749 this solution actually contained Ca concentrations of approximately 1-2 /M. We did not add chelating agents to the medium.) Text-fig. 2C shows a cell with a resting potential of -55 mV and an action potential in Ca-free medium with a 25 mV overshoot and a maximum rate of rise of 244 V/sec. In another test for a Ca component of the action potential, CoC12 (10 mM human, 5 mm gerbil), an agent known to block Ca spikes in other electrically active tissue (Hagiwara, 1973) was added to the medium. Action potentials were still recorded in the presence of Co (Text-fig. 2D). The hyperpolarizing after-potential seen in the chromaffin cells disappeared when the cells were hyperpolarized to approximately 70-100 mV and most likely represents an increased membrane conductance to potassium, as is seen in other excitable cells. A
B
C
D
E
Text-fig. 3. Chromaffin cell response to iontophoretically applied ACh. A, superimposed sweeps of hyperpolarizing and depolarizing current pulses (0-16 nA) applied through intracellular micro-electrode in chromaffin cell. Depolarizing pulse triggered an action potential. B, depolarizing potential produced by iontophoresis of ACh (I= 0-43 nA) on to chromaffin cell (at artifact). C, slightly larger iontophoresis current produced a larger depolarization and triggered an action potential identical to that seen in A. D, superimposed sweeps illustrating drop in membrane resistance (as measured by intracellular hyperpolarizing pulse) during ACh induced depolarization. E, three superimposed sweeps illustrating change in size of ACh induced depolarization (stimulus at arrow, -I=25 nA) with background depolarization and hyperpolarization of the chromaffin cell membrane. Calibration: 20 mV and 50 msec.
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Acetylcholine responsiveness Chromaffin cells are known to be sensitive to ACh both in situ and in vitro. It is the physiological stimulus for secretion. Kanno & Douglas (1967) showed that the application of ACh by a blunt micropipette depolarized chromaffin cells. We have applied ACh to the chromaffin cells by iontophoresis from fine-tipped micropipettes filled with 1 M-ACh and situated just outside the cell. Responses were found in most, but not all, cells. Text-fig. 3B illustrates a depolarizing potential evoked by ACh. The size of the depolarization was proportional to the current pulse used and depolarization did not occur if the polarity of the iontophoresis pulse was reversed. The response disappeared with small movements of the ACh pipette away from the cell. Text-fig. 30 demonstrates that the AChinduced depolarization could trigger an action potential identical to that seen with depolarizing current (trace 3A). It could be shown that ACh-induced depolarization was associated with a decreased resistance of the membrane as seen in Text-fig. 3D. In addition, the ACh-induced depolarization became larger during hyperpolarization of the chromaffin cell and smaller during depolarization of the chromaffin cell (Text-fig. 3E). Due to the inherent inaccuracies of measuring membrane potentials during passage of steady currents, it was not possible to determine an exact equilibrium potential for the depolarizing response. However, the response disappeared with background depolarizations to potentials less than approximately -20 mV. DISCUSSION
Our results clearly demonstrate that chromaffin cells in short term cultures and in adrenal slices are capable of generating neurone-like action potentials in response to depolarizing currents and in response to direct application of ACh. Our evidence further indicates that these action potentials are most likely generated by a Na mechanism with little or no direct calcium component. The possibility that neuronal contaminants are present in our cultures and could account for our results is very unlikely. Although there are neurones in the adrenal medulla, they comprise a small fraction of the total number of cells. The cells from which we recorded action potentials were numerous in culture, did not look like neuroses in culture, were identical to cells which showed a positive chromaffin reaction and catecholamine fluorescence, and, in two cases, were identified by electron microscopy as chromaffin cells. Therefore, we feel that the evidence supports the contention that the excitable cells were adrenal chromaffin cells.
CHROMA FFIN CELL ELECTRICAL EXCITABILITY 751 The experiments on the chromaffin cells of the adrenal medulla described in this paper were undertaken as a consequence of our previous investigation of the electrical properties of phaeochromocytomas (tumours derived from chromaffin cells) (Tischler, Dichter, Biales, DeLellis & Wolf, 1976). It was shown that the cells of five human and one established rat phaecochromocytoma develop neurone-like morphology in tissue culture and are capable of generating all-or-nothing overshooting action potentials. These cells may also show a depolarizing response to iontophoretically applied ACh. Similarly, cells from several other tumours (two human and one bovine medullary thyroid carcinoma, two human bronchial carcinoids and two human oat cell carcinomas have been shown to be electrically excitable. All of these tumours share with the chromaffin cells a proposed neural crest origin, but are not commonly described as neuroendocrine. Kidokoro (1975) has reported recording action potentials in a clonal pituitary cell line (GH3) and we have also recorded action potentials from two human pituitary adenomas in culture. Recently Davis & Hadley (1976) reported recording extracellular action potentials from cells in the neuro-intermediate lobe of the frog pituitary gland. One other endocrine tissue, the pancreatic fl-cell, has been shown to be electrically excitable (Dean & Mathews, 1968; Meissner & Schmelz, 1974) and the amount of fl-cell depolarization and action potential generation has been correlated with extracellular glucose concentration. A major question which arises as a consequence of our data in chromaffin cells is whether action potentials are normally involved in excitationsecretion coupling. ACh depolarizes chromaffin cells by causing an increased permeability to Na and possibly Ca and the influx of Ca is linked to the secretion of catecholamines (Douglas, 1975). By analogy with action potentials in nerve cells, it can be presumed that although Ca is not the major charge carrier during the action potential, small amounts of Ca will enter the cell during an action potential and this could be sufficient to link excitation with secretion. It has been shown that many means of depolarizing the chromaffin cell, even in the absence of external Na (a situation which should prevent action potential generation) can induce secretion as long as Ca is present in the medium (Douglas, 1975). However, the ability to demonstrate catecholamine secretion in response to nonphysiological stimuli under conditions which prevent action potential generation does not mean that action potentials are not involved under physiological conditions, since even neuronal synaptic terminals can be stimulated to secrete transmitter when action potentials are blocked. A more definitive experiment in the adrenal medulla would be to produce catecholamine secretion by a physiological stimulus in the presence of inhibitors of the chromaffin cell Na spike (i.e. TTX). However any agent
B. BIALES AND OTHERS which interferes with the chromaffin cell spike may equally affect the splanchnic nerve spike and make physiological stimulation impossible. At the present time, we can say that the chromaffin cell is capable of generating action potentials in response to applied ACh but the physiological significance of this response under normal conditions needs to be established. 752
This work was supported by a grant from the Esther A. and Joseph Klingenstein Foundation. Dr Dichter is supported by a USPHS-NINCDS Research Career Development Award 1 K04 NS 00130-01. Dr Tischler is supported by N.I.H. Grant Gm 568 for Training in Experimental Pathology and 1 RO Ca 17389-01. Our thanks to Mrs Sara Vasquez for technical support. REFERENCES COUPLAND, R. (1965). Electron microscopic observations on the structure of the rat adrenal medulla. I. The ultrastructure and organization of chromaffin cells in the normal adrenal medulla. J. Anat. 99, 231-254. DAVIS, M. & HADLEY, M. (1976). Spontaneous electrical potentials and pituitary hormone (MSH) secretion. Nature, Lond. 261, 422-423. DEAN, P. & MATHEWS, E. (1968). Electrical activity in pancreatic islet cells. Nature, Lond. 219, 389-390. DOUGLAS, W. (1975). Secrotomotor control of adrenal medullary secretion: synaptic, membrane and ionic events in stimulus-secretion coupling. In Handbook of Physiology, section 7, vol. 6, Endocrinology. Washington: American Physiological Society. DOUGLAS, W., KANNo, T. & SAMPSON, S. (1966). Intracellular recording from adrenal chromaffin cells: effects of acetylcholine, hexamethonium and potassium on membrane potentials. J. Physiol. 186, 125-126P. DOUGLAS, W., KANNo, T. & SAMPSON, S. (1967a). Effects of acetylcholine and other medullary secretagogues and antagonists on the membrane potential of adrenal chromaffin cells: an analysis employing techniques of tissue culture. J. Physiol. 188, 107-120. DouGLAs, W., KANNo, T. & SAMPSON, S. (1967b). Influence of the ionic environment on the membrane potential of adrenal chromaflin cells and on the depolarizing effect of acetylcholine. J. Phy8iol. 191, 107-121. DOUGLAS, W. & RUBIN, R. (1963). The mechanism of catecholamine release from the adrenal medulla and the role of calcium in stimulus secretion coupling. J. Physiol. 167, 288-310. FALcK, B., HIILARP, N.-A., THIEME, G. & ToRP, A. (1962). Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354. FAWCETT, P. (1969). An electrophysiological study of the rat adrenal gland in vivo. J. neurol. Scis 8, 381-383. HAGIWARA, S. (1973). Calcium spikes. Adv. Biophys. 4, 71-102. HILLARP, N.-A. & HOKFELT, B. (1955). Histochemical demonstration of adrenaline and noradrenaline in the adrenal medulla. J. Histochem. Cytochem. 3, 1-5. KANNO, T. & DoUGLAS, W. (1967). Effect of rapid application of acetylcholine or depolarizing current on transmembrane potentials of adrenal chromaffin cells. Proc. Can. Fedn Biol. Socs 10, 39. KIDOKORO, Y. (1975). Spontaneous calcium action potentials in a clonal pituitary cell line and their relationship to prolactin secretion. Nature. Lond. 258, 741-742.
Plate 1
The Journal of Physiology, Vol. 262, No. 3 r
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CIIROMAFFIN CELL ELECTRICAL EXCITABILITY 753 MATHEWS, E. (1967). Membrane potential measurement in cells of the adrenal gland. J. Phyiol. 189, 139-148. MEISSNER, H. & SCHMELZ, H. (1974). Membrane potential of beta-cells in pancreatic islets. Pfluger8 Arch. ges Physiol. 351, 195-206. NARAHASHI, T. (1974). Chemicals as tools in the study of excitable membranes. Physiol. Rev. 54, 812-889. TISCHLER, A., DICHTER, M., BIALEs, B., DELELLIS, R. & WOLFE, H. (1976). Neural properties of cultured human endocrine tumors of proposed neural crest origin. Science, N.Y. 192, 902-904. TISCHLER, A., DIC TER, M., BiALEs, B. & GREENE, L. (1976). Neuroendocrine neoplasms and their cells of origin. New Engl. J. Med. (in the Press). EXPLANATION OF PLATE
Human and gerbil chromaffin cells in culture. Phase contrast photomicrograph of penetrated human (A) and gerbil (B) chromaffin cells (bright cells) which demonstrated electrical excitability. C, single human chromaffin cell exhibiting formaldehyde induced fluorescence. This cell showed intense green fluorescence on a negative background. D, cluster of human chromaffin cells exhibiting a positive chromaffin reaction. The reaction product is brown. E, electron micrograph of gerbil chromaffin cell which had demonstrated an action potential with intracellular recording. Note the typical chromaffin granules. Calibration: A and B, 50 tm; E, 0-5 /sm.
743
ELECTRICAL EXCITABILITY OF CULTURED ADRENAL CHROMAFFIN CELLS
BY BERNARD BIALES, MARC DICHTER AND ARTHUR TISCHLER From the Departments of Neurology and Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.
(Received 10 May 1976) SUMMARY
1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro-electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded - 50 mV. 3. Chromaffin cells were capable of generating all-or-nothing overshooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 106 g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae. INTRODUCTION
The chromaffin cells of the mammalian adrenal medulla are catecholamine secreting cells derived from the neural crest. They receive a cholinergic preganglionic sympathetic innervation, mainly through the splanchnic nerve and are thus homologous with post-ganglionic sympathetic neurones. Acetylcholine (ACh) has been shown to stimulate catecholamine secretion in vivo (Douglas, 1975) and to depolarize chromaffin cells in vitro (Douglas, Kanno & Sampson, 1966, 1967 a, b). Notwithstanding the similarity of chromaffin cells to neurones, previous studies using microelectrode penetrations have found that chromaffin cells have low resting potentials both in vivo and in vitro and do not generate action potentials 26
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B. BIALES AND OTHERS (Douglas et al. 1967 a, b; Kanno & Douglas, 1967; Matthews, 1967; Fawcett, 1969). We have performed similar experiments on gerbil and human chromaffin cells in vitro and on the gerbil tissue slices. Our results demonstrate that chromaffin cells can maintain resting potentials greater than -50 mV and are capable of generating short duration all-or-nothing overshooting action potentials, both in response to direct electrical stimulation and to iontophoretic application of ACh. A preliminary report of these results was given at the 1975 Annual Meeting of the Society for Neuroscience. At the same meeting, Hagiwara & Kidokoro also reported findings of chromaffin cell excitability and ACh responsiveness. 744
METHODS Culture methods. For each dissociation, four to ten adult gerbil adrenal glands were aseptically excised, trimmed of fat, and sliced at the poles to allow access of enzymes. Sequential digestion was carried out under 95% air-5 % CO2 at 360 C in 2 ml. trypsin (Armour Pharmaceutical) and 1-2 ml. collagenase (Sigma) for 45-60 min each (both enzymes at 0.1 % in Eagle's MEM with 20 u. penicillin/ml. and 20 #sg streptomycin/ml., GIBCO). Between the two digestions the adrenal cortex was cut away and any mutilated medullae were discarded. After digestion, the tissue was placed in MEM with antibiotics, 200 mg % glucose and 10% rat serum (filtered through 20 jsm Nalgene filter and heat inactivated at 56° C for 30 min) and triturated successively through normal and flame-constricted Pasteur pipettes. Yield was typically 3000 cells per medulla. The cell suspension was spun down at 220 g for 4 min and resuspended in a small volume of the above medium plus 20-30 ng/ml. nerve growth factor (NGF) (kindly supplied by Dr Lloyd Greene) and 16 ,ug corticosterone/ml. (Sigma). The cells from j-2 medullae were plated in a volume of 0.1 ml. into a stainless-steel ring (4j mm i.d.) standing in a 35 mm plastic tissue culture dish (Falcon) containing 1-8 ml. medium outside the ring. The dishes were either untreated, coated with gelatin or collagen, or with the cells of a primary or secondary culture of rat embryonic lung previously treated with 10-5 M cytosine arabinoside to inhibit cell division. On the day following plating the restrictor ring was removed. Dishes were generally used within 3 days, but when kept beyond that, the medium was changed at 4 to 5day intervals. For experiments utilizing formaldehyde induced fluorescence, adrenal cells were plated on glass cover-slips previously coated with gelatin or lung cells. For human cultures, single adrenal glands were obtained from a 38 yr old man undergoing nephrectomy for hypertensive renal failure, a 57 yr old woman undergoing adrenalectomy for metastatic breast carcinoma (histological examination revealed no metastatic lesions) and a 61 yr old man with Cushing's disease. Dissociation and plating were carried out as described above except that the medullary regions were excised before trypsinization, digestion periods were extended to 75 min each and the inoculum was raised to 9000-27,000 cells. For recording from adrenal slices, adult gerbil adrenal medullae were excised, dividedand wedged, cut side up, in a block of saline-agar. Penetrations were guided
by eye. Electrophysiology. Micro-electrode penetrations were made in physiological saline (Na 147, Cl 153, K 3 4, Ca 1-8, Mg 1.0, H2PO4 0 4, HPO4 2-4, glucose 8 mm, pH 7.3) with phase contrast visualization at 31-35° C. To allow the addition of Co to the
CHROMAFFIN CELL ELECTRICAL EXCITABILITY
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medium, a similar Tris (2-5 or 5-0 mm at pH 7-2) buffered saline without phosphate was used. Micro-electrodes were made from Corning 7740 glass tubing (o.d. 1 mm, i.d. 0-8 mm) using a modified Kopf vertical puller. They were first filled with double distilled water by boiling for 5-12 min, then back-filled with 3-5 N-K acetate, using a 30-gauge needle. Electrodes were allowed to equilibrate for at least 11 hr before use. Best results were obtained with 40-120 MO electrodes. ACh chloride (1 M, Sigma) electrodes were made in the same manner. Electrodes were connected to an electrometer amplifier (WPI M701 or M4A) by a chlorided Ag wire. Current was carried to ground through an agar-saline bridge to a Ag-AgCl electrode in a 3 N-KC1 bath. Output from the amplifier was displayed on a dual beam oscilloscope (Tektronix) and a multichannel chart recorder (Gould 260). Provision was made for simultaneously passing steady currents and square-wave pulses through the recording micro-electrode by way of a circuit built into the WPI pre-amplifier. The cells were stimulated by either a 40-50 msec square-wave of depolarizing current injected into the cell or by a 0-5 msec pulse injected into the cell (Text-fig. 1 D, E). For ACh iontophoresis, ACh-filled micro-electrodes (tip 0-5 lam, R =100-200 MO) were positioned under microscopic control to within 5 #sm of the cell being stimulated. Most electrodes required a hyperpolarizing 'holding' current of between 1 and 5 nA to prevent depolarization as the electrode approached the cell. Iontophoretic responses were sought with short (1-4 msec), 0-2-25 nA depolarizing pulses superimposed on the steady 'holding' current. Current was monitored through the WPI pre-amplifier. (ell identification. The chromaffin reaction was performed on cultures fixed for 10-14 hr, in a solution of K2Cr2O4 and K2CrO4 according to the method of Hillarp & Hokfelt (1955). For formaldehyde induced fluorescence, cover-slip cultures of adrenal medulla cells were rapidly blown dry at room temperature and then further air dried over Pr05 for at least 3 days. They were then heated at 800 C for 3 hr over paraformaldehyde which had been equilibrated at 60 % humidity according to the method of Falck, Hillarp, Thieme & Torp (1962). The cover-slips were examined with an AO fluorescence microscope equipped with a BG12 excitor and 470 barrier filter. Electron micro8copy. Cultures were fixed in 3% glutaraldehyde (TAAB), pH 7 3, in tissue culture medium and post-fixed with 1 % osmium in 0-1 phosphate buffer at pH 7-0. Ultrathin sections (80-100 nm) were stained with uranyl acetate and lead citrate. RESULTS
Cell morphology and identification in culture Living chromaffin cells could be recognized in culture by phase contrast microscopy (PI. 1A, B). Single cells initially appeared as spheres (about 8-30 ,sm diameter), but later often flattened or assumed a spindle shape. They had finely granulated cytoplasm with a medium sized reticulated nucleus and small nucleolus. Cells were often seen in clusters and although groups of cells had a characteristic range of morphology, it was sometimes difficult to pick out the boundary of individual cells in the clusters. The identification of the chromaffin cells in vitro was established by a variety of histological techniques. The cells showed intense green fluorescence following treatment with formaldehyde vapour (P1. 1 C), indicating catecholamine content. The cells also exhibited a positive
746 B. BIALES AND OTHERS chromaffinreaction (P1. ID). Finally, electron microscopy of two cellswhich were penetratedwith micro-electrodes and which exhibited action potentials (see below) demonstrated large (typically 0-2 mcm) dense core granules characteristic of chromaffin cells (Coupland, 1965) (PI. 1E).
Electrophysiology Results of intracellular recordings in both gerbil and human cultures were similar. A few cells had membrane resting potentials of -50 to -70 mV, usually attained after spontaneous improvement from an initially lower potential. These cells exhibited all-or-nothing, overshooting action potentials either spontaneously or in response to membrane depolarization (Text-fig. 1A-E). This most active group of cells A
D
C
c
\
E
F.
''
Text-fig. 1. Chromaffin cell action potentials. A, human cell in culture. B, gerbil cell in culture. C, gerbil adrenal medulla slice with one subthreshold and one suprathreshold depolarizing pulse. D, action potential evoked by short depolarizing pulse. E, action potential as in D but with swept out trace. F, repetitive firing with prolonged depolarizing pulse. Calibration: 10 mV and 5 msec (except 0 5 msec for E). Stimulus currents: A, 0-33 nA; B, 0-14 nA; C, 0-28 nA; F, 1-25 nA.
also showed repetitive firing when depolarized beyond threshold (Textfig. iF). More commonly, the chromaffin cells had resting potentials in the range of -10 to -40 mV and action potentials could be elicited by depolarization subsequent to background hyperpolarization of the cells. In the most active cells, rates of rise of the action potential reached 100-220 V/sec and average spike duration at half-height was 2 msec. The action potentials were followed by a hyperpolarizing after-potential. Gerbil chromaffin cells maintained in the presence of NGF (10 ng/ml.),
CHROMAFFIN CELL ELECTRICAL EXCITABILITY 747 corticostorone (17 mcg/ml.), NGF and corticosterone, or without either additive, all demonstrated similar action potentials. In order to be certain that the ability to generate action potentials was not an artifact induced by maintaining the chromaffin cells in vitro, we recorded from chromaffin cells in slices of freshly isolated adult gerbil adrenals. Penetrations were more difficult than in vitro but we were able to observe numerous chromaffin cells which were capable of generating action potentials (Text-fig. 1 C). We cannot comment on mean resting potentials of the cells from which we recorded for several reasons. The cells were relatively small and fragile and most showed extensive signs of injury after penetration - either morphological or electrophysiological. In addition we used relatively high resistance micro-electrodes (up to 150 MD) with variable, and sometimes high, tip potentials. However, it was clear that at least some cells were capable of sustaining high resting potentials. We cannot tell from our data whether the relatively low values for resting potentials which we often obtained and which others have reported (Douglas et al. 1967a, b; Matthews, 1967; Fawcett, 1969) represent the true situation in sitn or are artifacts due to penetration injury. We suspect the latter. One reason for this suspicion is the fact that so many of our 'poor' cells which initially appeared inexcitable could be induced to fire action potentials by background hyperpolarization. Induction of such action potentials has never been reported as an artifact and does not occur in many other nonexcitable tissues. For similar reasons we have not specified the percentage of chromaffin cells which could generate action potentials. These data are meaningless if they include all the injured cells. Although we cannot rule out the possibility that some cells are inexcitable, in four experiments where we tried to deal with this question, thirty out of thirty sequentially penetrated human cells and fourteen out of fifteen gerbil cells could be induced to fire action potentials. In total we recorded from well over 300 electrically active chromaffin cells. Ionic basis of the action potential The chromaffin cell action potential disappeared in tetrodotoxin (TTX) at 10-7 to 10-6 g/ml. TTX is known to block most Na spike mechanisms (Narahashi, 1974). These experiments were performed by sequentially penetrating different cells in control medium, TTX-medium, and back in control (after four washes). In every case it immediately became impossible for the chromaffin cells to generate action potentials in the presence of TTX. (In a few cells in TTX, 10-7 g/ml., there were small (ca. 10 mV) potentials which occurred on top of the passive depolarizing charging curve. It was difficult to distinguish these from delayed
748 B. BIALES AND OTHERS rectification. These small extra potentials were not present in TTX, 10-6 g/ml.) After washing the chromaffin cells with control medium the cells were again regularly able to generate action potentials. Text-fig. 2A is an example of a human chromaffin cell with a resting potential of -30 mV and an input resistance of approximately 90 MQ which did not show an active response in TTX (t0-7 g/ml.) despite background hyperpolarization. Text-fig. 2 B shows a re-penetration of the same A
B
c
D
Text-fig. 2. Ionic basis of chromaffin cell action potential. A, large depolarizing pulses produce no active response in presence of tetrodotoxin (10-6 g/ml.). B, same cell as in A after removing TTX. Resting membrane potential and input resistance were lower but cell could still generate action potentials. C, repetitive spikes from chromaffin cell in Ca-free medium. Upper trace indictes zero base line and duration of intracellular current pulse. D, chromaffin cell action potential in presence of CoCl2 (10 mm). Calibration: 50 mV and 25 msec for all traces. Stimulus currents: A, 1-8, 2-5 nA; B, 1-1 nA; C, 0-21 nA; D, 0-24 nA.
cell in control which then showed somewhat inferior resting membrane parameters (low resting potential and input resistance approximately 50 MQ) but which could generate all-or-nothing action potentials. The above results indicate that the chromaffin cell action potentials are most likely generated by a Na mechanism. Since Ca ions have been shown to be necessary for adrenal medullary secretion (Douglas, 1975), we attempted to determine if Ca ions were necessary for action potential generation. In medium to which no Ca had been added, the chromaffin cells were still capable of generating large action potentials. (Although we will refer to this as 'Ca-free' medium,
CHROMAFFIN CELL ELECTRICAL EXCITABILITY 749 this solution actually contained Ca concentrations of approximately 1-2 /M. We did not add chelating agents to the medium.) Text-fig. 2C shows a cell with a resting potential of -55 mV and an action potential in Ca-free medium with a 25 mV overshoot and a maximum rate of rise of 244 V/sec. In another test for a Ca component of the action potential, CoC12 (10 mM human, 5 mm gerbil), an agent known to block Ca spikes in other electrically active tissue (Hagiwara, 1973) was added to the medium. Action potentials were still recorded in the presence of Co (Text-fig. 2D). The hyperpolarizing after-potential seen in the chromaffin cells disappeared when the cells were hyperpolarized to approximately 70-100 mV and most likely represents an increased membrane conductance to potassium, as is seen in other excitable cells. A
B
C
D
E
Text-fig. 3. Chromaffin cell response to iontophoretically applied ACh. A, superimposed sweeps of hyperpolarizing and depolarizing current pulses (0-16 nA) applied through intracellular micro-electrode in chromaffin cell. Depolarizing pulse triggered an action potential. B, depolarizing potential produced by iontophoresis of ACh (I= 0-43 nA) on to chromaffin cell (at artifact). C, slightly larger iontophoresis current produced a larger depolarization and triggered an action potential identical to that seen in A. D, superimposed sweeps illustrating drop in membrane resistance (as measured by intracellular hyperpolarizing pulse) during ACh induced depolarization. E, three superimposed sweeps illustrating change in size of ACh induced depolarization (stimulus at arrow, -I=25 nA) with background depolarization and hyperpolarization of the chromaffin cell membrane. Calibration: 20 mV and 50 msec.
750
B. BIALES AND OTHERS
Acetylcholine responsiveness Chromaffin cells are known to be sensitive to ACh both in situ and in vitro. It is the physiological stimulus for secretion. Kanno & Douglas (1967) showed that the application of ACh by a blunt micropipette depolarized chromaffin cells. We have applied ACh to the chromaffin cells by iontophoresis from fine-tipped micropipettes filled with 1 M-ACh and situated just outside the cell. Responses were found in most, but not all, cells. Text-fig. 3B illustrates a depolarizing potential evoked by ACh. The size of the depolarization was proportional to the current pulse used and depolarization did not occur if the polarity of the iontophoresis pulse was reversed. The response disappeared with small movements of the ACh pipette away from the cell. Text-fig. 30 demonstrates that the AChinduced depolarization could trigger an action potential identical to that seen with depolarizing current (trace 3A). It could be shown that ACh-induced depolarization was associated with a decreased resistance of the membrane as seen in Text-fig. 3D. In addition, the ACh-induced depolarization became larger during hyperpolarization of the chromaffin cell and smaller during depolarization of the chromaffin cell (Text-fig. 3E). Due to the inherent inaccuracies of measuring membrane potentials during passage of steady currents, it was not possible to determine an exact equilibrium potential for the depolarizing response. However, the response disappeared with background depolarizations to potentials less than approximately -20 mV. DISCUSSION
Our results clearly demonstrate that chromaffin cells in short term cultures and in adrenal slices are capable of generating neurone-like action potentials in response to depolarizing currents and in response to direct application of ACh. Our evidence further indicates that these action potentials are most likely generated by a Na mechanism with little or no direct calcium component. The possibility that neuronal contaminants are present in our cultures and could account for our results is very unlikely. Although there are neurones in the adrenal medulla, they comprise a small fraction of the total number of cells. The cells from which we recorded action potentials were numerous in culture, did not look like neuroses in culture, were identical to cells which showed a positive chromaffin reaction and catecholamine fluorescence, and, in two cases, were identified by electron microscopy as chromaffin cells. Therefore, we feel that the evidence supports the contention that the excitable cells were adrenal chromaffin cells.
CHROMA FFIN CELL ELECTRICAL EXCITABILITY 751 The experiments on the chromaffin cells of the adrenal medulla described in this paper were undertaken as a consequence of our previous investigation of the electrical properties of phaeochromocytomas (tumours derived from chromaffin cells) (Tischler, Dichter, Biales, DeLellis & Wolf, 1976). It was shown that the cells of five human and one established rat phaecochromocytoma develop neurone-like morphology in tissue culture and are capable of generating all-or-nothing overshooting action potentials. These cells may also show a depolarizing response to iontophoretically applied ACh. Similarly, cells from several other tumours (two human and one bovine medullary thyroid carcinoma, two human bronchial carcinoids and two human oat cell carcinomas have been shown to be electrically excitable. All of these tumours share with the chromaffin cells a proposed neural crest origin, but are not commonly described as neuroendocrine. Kidokoro (1975) has reported recording action potentials in a clonal pituitary cell line (GH3) and we have also recorded action potentials from two human pituitary adenomas in culture. Recently Davis & Hadley (1976) reported recording extracellular action potentials from cells in the neuro-intermediate lobe of the frog pituitary gland. One other endocrine tissue, the pancreatic fl-cell, has been shown to be electrically excitable (Dean & Mathews, 1968; Meissner & Schmelz, 1974) and the amount of fl-cell depolarization and action potential generation has been correlated with extracellular glucose concentration. A major question which arises as a consequence of our data in chromaffin cells is whether action potentials are normally involved in excitationsecretion coupling. ACh depolarizes chromaffin cells by causing an increased permeability to Na and possibly Ca and the influx of Ca is linked to the secretion of catecholamines (Douglas, 1975). By analogy with action potentials in nerve cells, it can be presumed that although Ca is not the major charge carrier during the action potential, small amounts of Ca will enter the cell during an action potential and this could be sufficient to link excitation with secretion. It has been shown that many means of depolarizing the chromaffin cell, even in the absence of external Na (a situation which should prevent action potential generation) can induce secretion as long as Ca is present in the medium (Douglas, 1975). However, the ability to demonstrate catecholamine secretion in response to nonphysiological stimuli under conditions which prevent action potential generation does not mean that action potentials are not involved under physiological conditions, since even neuronal synaptic terminals can be stimulated to secrete transmitter when action potentials are blocked. A more definitive experiment in the adrenal medulla would be to produce catecholamine secretion by a physiological stimulus in the presence of inhibitors of the chromaffin cell Na spike (i.e. TTX). However any agent
B. BIALES AND OTHERS which interferes with the chromaffin cell spike may equally affect the splanchnic nerve spike and make physiological stimulation impossible. At the present time, we can say that the chromaffin cell is capable of generating action potentials in response to applied ACh but the physiological significance of this response under normal conditions needs to be established. 752
This work was supported by a grant from the Esther A. and Joseph Klingenstein Foundation. Dr Dichter is supported by a USPHS-NINCDS Research Career Development Award 1 K04 NS 00130-01. Dr Tischler is supported by N.I.H. Grant Gm 568 for Training in Experimental Pathology and 1 RO Ca 17389-01. Our thanks to Mrs Sara Vasquez for technical support. REFERENCES COUPLAND, R. (1965). Electron microscopic observations on the structure of the rat adrenal medulla. I. The ultrastructure and organization of chromaffin cells in the normal adrenal medulla. J. Anat. 99, 231-254. DAVIS, M. & HADLEY, M. (1976). Spontaneous electrical potentials and pituitary hormone (MSH) secretion. Nature, Lond. 261, 422-423. DEAN, P. & MATHEWS, E. (1968). Electrical activity in pancreatic islet cells. Nature, Lond. 219, 389-390. DOUGLAS, W. (1975). Secrotomotor control of adrenal medullary secretion: synaptic, membrane and ionic events in stimulus-secretion coupling. In Handbook of Physiology, section 7, vol. 6, Endocrinology. Washington: American Physiological Society. DOUGLAS, W., KANNo, T. & SAMPSON, S. (1966). Intracellular recording from adrenal chromaffin cells: effects of acetylcholine, hexamethonium and potassium on membrane potentials. J. Physiol. 186, 125-126P. DOUGLAS, W., KANNo, T. & SAMPSON, S. (1967a). Effects of acetylcholine and other medullary secretagogues and antagonists on the membrane potential of adrenal chromaffin cells: an analysis employing techniques of tissue culture. J. Physiol. 188, 107-120. DouGLAs, W., KANNo, T. & SAMPSON, S. (1967b). Influence of the ionic environment on the membrane potential of adrenal chromaflin cells and on the depolarizing effect of acetylcholine. J. Phy8iol. 191, 107-121. DOUGLAS, W. & RUBIN, R. (1963). The mechanism of catecholamine release from the adrenal medulla and the role of calcium in stimulus secretion coupling. J. Physiol. 167, 288-310. FALcK, B., HIILARP, N.-A., THIEME, G. & ToRP, A. (1962). Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354. FAWCETT, P. (1969). An electrophysiological study of the rat adrenal gland in vivo. J. neurol. Scis 8, 381-383. HAGIWARA, S. (1973). Calcium spikes. Adv. Biophys. 4, 71-102. HILLARP, N.-A. & HOKFELT, B. (1955). Histochemical demonstration of adrenaline and noradrenaline in the adrenal medulla. J. Histochem. Cytochem. 3, 1-5. KANNO, T. & DoUGLAS, W. (1967). Effect of rapid application of acetylcholine or depolarizing current on transmembrane potentials of adrenal chromaffin cells. Proc. Can. Fedn Biol. Socs 10, 39. KIDOKORO, Y. (1975). Spontaneous calcium action potentials in a clonal pituitary cell line and their relationship to prolactin secretion. Nature. Lond. 258, 741-742.
Plate 1
The Journal of Physiology, Vol. 262, No. 3 r
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BERNARD BIALES
AND OTHERS
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CIIROMAFFIN CELL ELECTRICAL EXCITABILITY 753 MATHEWS, E. (1967). Membrane potential measurement in cells of the adrenal gland. J. Phyiol. 189, 139-148. MEISSNER, H. & SCHMELZ, H. (1974). Membrane potential of beta-cells in pancreatic islets. Pfluger8 Arch. ges Physiol. 351, 195-206. NARAHASHI, T. (1974). Chemicals as tools in the study of excitable membranes. Physiol. Rev. 54, 812-889. TISCHLER, A., DICHTER, M., BIALEs, B., DELELLIS, R. & WOLFE, H. (1976). Neural properties of cultured human endocrine tumors of proposed neural crest origin. Science, N.Y. 192, 902-904. TISCHLER, A., DIC TER, M., BiALEs, B. & GREENE, L. (1976). Neuroendocrine neoplasms and their cells of origin. New Engl. J. Med. (in the Press). EXPLANATION OF PLATE
Human and gerbil chromaffin cells in culture. Phase contrast photomicrograph of penetrated human (A) and gerbil (B) chromaffin cells (bright cells) which demonstrated electrical excitability. C, single human chromaffin cell exhibiting formaldehyde induced fluorescence. This cell showed intense green fluorescence on a negative background. D, cluster of human chromaffin cells exhibiting a positive chromaffin reaction. The reaction product is brown. E, electron micrograph of gerbil chromaffin cell which had demonstrated an action potential with intracellular recording. Note the typical chromaffin granules. Calibration: A and B, 50 tm; E, 0-5 /sm.
