Stimulus-Secretion Coupling in Chromaffin Cells Isolated

Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 5036-5040, November 1977 Cell Biology

Stimulus-secretion coupling in chromaffin cells isolated from bovine adrenal medulla (catecholamine secretion/acetylcholine receptors/K+ depolarization/calcium requirement/microtubule disruption) ALLAN S. SCHNEIDER*, RUTH HERZ*, AND KURT ROSENHECKt * Sloan-Kettering Institute for Cancer Research, Cornell University Graduate School of Medical Sciences, New York, New York 10021; and tDepartment of Membrane Research, Weizmann Institute of Science, Rehovot, Israel Communicated by Francis 0. Schmitt, August 19,1977

ABSTRACT Bovine adrenal chromaffin cells were isolated membrane fusion and exocytosis, (iii) the possible role of cyclic by removal of the cortex and sequential collagenase digestion nucleotides coupled with ion translocation in mediating hor- of the medulla. The catecholamine secretory function of these cells was characterized with respect to acetylcholine stimula- mone and neurotransmitter synthesis and secretion, and (iv) tion, cation requirements, and cytoskeletal elements. The the role of microfilaments, microtubules, and/or a calcium- dose-response curve for stimulated release had its half-maxi- modulated contractile protein system in bringing secretory mum value at 10-5 M acetylcholine, and maximum secretion granules together with the plasma membrane to facilitate ex- was on the average 7 times that of control basal secretion. The ocytosis. Further advances may be possible on these and related differential release of epinephrine versus norepinephrine after questions through the use of isolated chromaffin cells. stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic re- In the present work we provide a functional characterization ceptors were found to be predominantly nicotinic. The kinetics of bovine adrenal chromaffin cells isolated in large quantities of catecholamine release were rapid, with significant secretion (107-108 cells) and essentially free of cortical cells. We report occurring in less than 60 sec and 85% of maximum secretion the dependence of the secretory function of these cells on time, within 5 min. A critical requirement for calcium in the extra- ACh concentration, potassium depolarization, calcium con- cellular medium was demonstrated, and 80% of maximum se- centration, and nicotinic and muscarinic agents. In addition, cretion was achieved at physiologic calcium concentrations. Stimulation by excess potassium (65 mM KCI) also induced data are presented on the differential release of epinephrine catecholamine secretion which differed from acetylcholine and norepinephrine and on the secretory effects of microtubule- stimulation in being less potent, in having a different depen- and microfilament-disrupting agents and of cholinesterase dence on calcium concentration, and in its response to the local inhibitors. anesthetic tetracaine. Tetracaine, which is thought to inhibit Isolated adrenal chromaffin cells were first used by Douglas membrane cation permeability, was able to block acetylcho- and coworkers (5, 6) in mixed tissue culture to study their line-stimulated but not KCI-stimulated secretion. The microtubule disrupting agent vinblastine was able to block cate- electrophysiological properties by microelectrode techniques. cholamine release whereas the microfilament disrupter cyto- More recently, other workers have reported isolation of adrenal chalasin B had little effect. The results show the isolated bovine cells (7-9). Brandt et al. (7) and Biales et al. (8) discovered chromaffin cells to be viable, functioning, and available in large transmembrane action potentials in isolated chromaffin cells quantity. These cells now provide an excellent system for in tissue culture, the former workers using rat and the latter both studying cell surface regulation of hormone and neuro- gerbil and human chromaffin cells. Hochman and Perlman (9) transmitter release. isolated a mixture of chromaffin and cortical cells from whole The term "stimulus-secretion coupling" was originally coined guinea pig adrenal glands and used these to study the conditions by Douglas and Rubin (1, 2) more than a decade ago to describe of catecholamine secretion. In the above studies with rodent the sequence of events initiated by acetylcholine (ACh) stim- adrenal glands, relatively small quantities of chromaffin cells ulation of adrenal chromaffin cells and leading to secretion of were obtained per gland (103-105) due to the small gland catecholamines by exocytosis. They had in mind the close size. similarity to the phenomenon of "excitation-contraction cou- There are several advantages of the isolated bovine chro- pling" in muscle (namely, the key role of calcium in mediating maffin cell system that should prove useful in future studies. both secretion and contraction and a parallel set of electrical First, the system is relatively well defined and available in large and ionic events at the plasma membrane in response to ACh). quantity. Second, the absence of cortical cells should aid future Much progress has been made during the past 2 decades in studies of both the cyclic nucleotide response and differential elucidating the various steps in the mechanism of secretion, with release, because cortical cells are known to have their own cyclic a substantial portion of the data deriving from studies on the nucleotide system and corticosteroids are known to affect the perfused adrenal gland (2-4). There remain, however, many conversion of norepinephrine to epinephrine. Third, the kinetics surface of secretion of coupling between cholinergic stimulation and the secretory important aspects of cell receptor regulation response should be more easily resolved in the isolated cell that are still poorly understood, such as (i) the molecular nature in kinetics of of the calcium permeability sites that are altered by activated suspension than the perfused gland, because the cholinergic receptors and potassium depolarization, (ii) the perfusion and tissue penetration are not complicating factors. which calcium into the cell induces Fourth, isolated cell suspensions offer the possibility of spec- mechanism by entry trofluorometric study of membrane fluidity, ion translocation; receptor topography, etc., by using appropriate fluorescent The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked probes. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: ACh, acetylcholine. 5036 Downloaded by guest on September 27, 2021 Cell Biology: Schneider et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5037

Table 1. Catecholamine' content and differential release 100 0 from isolated chromaffin cells * ACh (10-4M) Total catecholamines, 80/ z nmol/mg of protein % 0 Mean Range epinephrine LUJ 60_ Adrenal medulla* 577 (529-685) 75 LU(0. Cells (day W)t 347 (221-696) 46 U-I) Cells (day 2)t 332 (195-769) 42 40 / ACh (0.1 mM) stimulatedt secretion 72 ( 21-136) 42 20 * Control * Six independent assays on four excised medullas. t Thirteen independent cell preparations. Twenty-seven independent supernatants of stimulated cells at 370, C 20 min, 2 mM CaCl2. Mean (±SD) stimulated release was 7.0 + 3.7 0 4 8 12 16 20 times control basal secretion. TIME (minutes) FIG. 1. Kinetics of catecholamine secretion. Upper curve, 0.1 mM MATERIALS AND METHODS ACh in buffer II; lower curve, control basal secretion in buffer II. Maximum secretion = 74 nmol/mg of protein = 6.4 1.9 (SD) X Cell Preparation. Adrenal glands were removed from bulls control. immediately after slaughter, placed on ice, and delivered to the laboratory within 60-90 min. The chromaffin cell isolation sion intensities of epinephrine and norepinephrine standards procedure was a modification of that reported by Kloppenberg were approximately equal at equal concentrations in the range et al. (10) and Hochman and Perlman (9) for isolation of of 1-10 nmol. For differential determination, oxidations were adrenal cells from whole adrenal glands. In our procedure, the carried out at pH 3.5 (excitation, 410 nm; emission, 520 rm) and cortex, which is easily distinguished from the medulla by the pH 6.0 (excitation, 400 nm; emission, 510 nm). Fluorescence unaided eye, was removed and discarded. The medulla was was monitored with a Perkin-Elmer MPF4 spectrofluorome- then cut into fine pieces (-1 mm) and rinsed thoroughly in cold ter. Ca2+-free Krebs-Ringer bicarbonate glucose buffer, pH 7.2-7.4 We compared the results of the catecholamine assay with and (buffer I, ref. 9). Six to eight medullas were processed at a time without a prior alumina adsorption as described by Anton and (total wet weight, 15-25 g for the cut and washed pieces). The Sayre (14). The effect of alumina adsorption was relatively small cells were separated from the tissue by four or five sequential for medullas (11% increase) and supernatants containing se- collagenase digestions (Type I, Worthington Biochemicals, 0.2% creted catecholamines (6% increase) and slightly larger for cells in buffer 1, 2 ml/g wet weight), each of 30-min duration at 370 (24% increase). Secreted catecholamines were routinely assayed under 95% 02/5% CO2. Cells were harvested after each di- without prior alumina adsorption. gestion by filtration; the first harvest was discarded because it contained damaged cells and erythrocytes. Subsequent cell RESULTS harvests were washed with the same buffer supplemented with The data are generally reported as % maximum secretion, with 2.0 mM CaCl2 and 0.5% bovine serum albumin (buffer II) and the latter representing secretion under conditions of 2 mM Ca2+ finally pooled. Pooled cells were washed at least two additional and 0.1 mM ACh unless otherwise indicated. The amount of times by centrifugation at 110 X g for 5 min, filtered through catecholamines present in the extracellular medium before a 100-,um acetate mesh, and diluted to a final concentration of stimulation experiment was begun (due to damaged cells) was I to 2 X 107 cells per ml. Viability as measured by trypan blue subtracted from the total released catecholamines. or erythrosine B exclusion tests was 80-90%. Protein in cells Table 1 shows the mean values of the total and differential washed free of contamination from the medium was deter- (% epinephrine) catecholamine content of bovine adrenal mined by the method of Lowry et al. (11). Cells were used fresh medulla, isolated chromaffin cells, and 0.1 mM ACh-stimulated or stored overnight in buffer II at room temperature with 100 secretion. The mean (±SD) stimulated secretion at 0.1 mM ACh units of penicillin and 100 ,ug of streptomycin per ml. On the and 2.0 mM Ca2+ was 7.0 + 3.7 times that of control basal levels second day the cells were washed before use; viability was of secretion in buffer II at 37°. The differential catecholamine 80-85%. content of the bovine adrenal medulla was 75% epinephrine Stimulation of Catecholamine Release. To monitor cellular and 25% norepinephrine, in good agreement with earlier de- secretory response, 1 to 2 X 105 cells per ml in a total volume terminations (15, 16). The isolated cells were found to have a of 1-2 ml of buffer II were incubated, with various agents af- lower epinephrine content than the medulla, and this may be fecting secretion, in plastic tubes in a shaking water bath at 370 due to increased lability of the epinephrine-containing cells for 20 min. When 65 mM K+ was used, buffer II was modified during tissue disaggregation. The proportion of epinephrine so that the Na+ content was decreased to maintain isotonicity. in the ACh-stimulated secretion was about the same as that in To stop release, tubes were placed in an ice-water bath, then the cells. centrifuged for 5 min at 200 X g in the cold. A measured por- The kinetics of release are shown in Fig. 1 for cells stimulated tion of supernatant was drawn off, added to perchloric acid to with 0.1 mM ACh and for unstimulated control cells repre- give a final concentration of 0.4 M perchloric acid, and frozen senting basal levels of secretion. Calcium (2 mM) was present until assayed for catecholamine content. in both cases. The secretory response to ACh was quite rapid Catecholamine Assay. Catecholamine was measured by the with significant release occurring within 1 min (20% maximum) trihydroxyindole method of Euler and Floding (12) with and 85% of maximum secretion occurred within 5 min. modifications (13). For total catecholamines, excitation A dose-response curve is shown in Fig. 2 for ACh-stimulated wavelengths between 395 and 410 nm and emission wave- secretion. The half-maximum secretion occurred at an ACh lengths between 500 and 515 nm were chosen such that emis- concentration of 10 ,uM and maximum secretion, between 50 Downloaded by guest on September 27, 2021 5038 Cell Biology: Schneider et al. Proc. Nati. Acad. Sci. USA 74 (1977)

z 2 80_ LU Z 0 LU 60_ 0 a: 60 0 6 E 40/ Cr0,K6 M

D 20_ 40 0~~~~~~~~~~~~~~~~~~~~~~~1~ 1°46 10-5 10-4 IU:3 ACETYLCHOLINE (M) 0--20- FIG. 2. Dose-response curve for ACh stimulation of catecholamine secretion. Maximum secretion = 70 nmol/mg of protein = 8.0 0 2 4 6 8 1 4.0 (SD) X control. CALCIUM (mM) FIG. 3. Calcium dependence of secretion stimulated by 0.1 mM ,uM and 0.1 mM ACh. A slight decrease in catecholamine se- ACh (upper curve) or by excess potassium'(65 mM) (lower curve). cretion was consistently found at higher ACh concentrations Maximum secretion (at 0.1 mM ACh and 8 mM Ca2+) = 102 nmol/mg to 1 mM. of protein = 5.6 1 2.9 (SD) X control. In order to test for a possible effect of acetylcholinesterase on cholinergic stimulated secretion, we added known cholin- obtained by maximal ACh stimulation. We explored higher KC1 esterase inhibitors (eserine and neostigmine) to the stimulation concentrations, up to 100 mM, without significant increase in assay. In addition, the secretory response to a nondegradable secretory response at 2 mM Ca2+. Tetracaine (0.5 mM) was able ACh analog, carbachol, was compared with that to ACh (Table to block about 70% of the ACh-stimulated release but had little 2). No enhancement was found with carbachol in a concen- effect on the K+-stimulated release. Tetracaine alone resulted tration range 10 AM to 1 mM compared with ACh at 10MM and in a small (30% maximum secretion) but reproducible effect 0.1 mM. Addition of eserine to 10 MM ACh did not enhance which, if subtracted from the ACh + tetracaine value, would secretion, but a small although possibly insignificant en- indicate a total tetracaine block of ACh-induced secretion. hancement was found with addition of neostigmine. These data The nature of the cell surface cholinergic receptors was do not suggest any substantive acetylcholinesterase activity on further characterized with respect to nicotinic and muscarinic the surface of the isolated bovine chromaffin cells, and similar agents (Fig. 5). The major secretory response was found with results have been noted with adrenal cells isolated from the nicotine; only a small and possibly insignificant response was guinea pig (9). obtained with muscarine (1.0-0.01 mM) and the muscarinic In Fig. 3 we show the calcium requirement for secretion with agonist pilocarpine (1.0-0.01 mM). The ACh-stimulated se- either 0.1 mM ACh or excess KCl (65 mM) as secretagogue. cretion was blocked by the nicotinic antagonist hexamethoni- There was an absolute requirement for calcium (i.e., no de- um. tectable secretion at concentrations below 20MM Ca2+). Second, There have been suggestions in the literature of possible in- there was an obvious difference in calcium dependence of se- volvement of cytoskeletal elements in the mechanism of cretory function between ACh stimulation and KCI stimulation. catecholamine secretion (17, 18). We tested the effects of vin- The ACh curve rises abruptly to about 80% of maximum se- blastine and cytochalasin B, known disrupters of microtubules cretion at physiologic calcium concentrations (-2 mM) and and microfilaments, respectively, on ACh-stimulated cate- then levels off to a very slight rise with increasing calcium. The cholamine secretion (Fig. 6). Vinblastine at 50 ,M decreased KCI stimulated secretion is lower and does not show the leveling ACh-stimulated secretion to 23%, which is close to the effect off but instead continues to rise approximately linearly with increasing calcium concentration up to 8 mM. Above 8 mM, 100 _ the cells began to aggregate and no higher calcium concentrations were explored. 80_ Fig. 4 compares the effects of the local anesthetic tetracaine z on secretion stimulated by 0.1 mM ACh and 65 mM KC1. KC1 0 _ depolarization gives about 55% of the catecholamine release LU u 60_ Table 2. Effect of acetylcholinesterase inhibitors Ln and carbachol on secretion 2 _ 2 40_ % maximum secretion 2 0 ACh, 0.1 mM 100* 20 ACh, 10MM 55:: 13 ACh (10 MM) + eserine (10 MM) 59 4 10 ACh (10 MM) + neostigmine (10 ,M) 67 i 9 ACh ACh K0(65mM) 0(65mM) TETRA Carbachol, 10MLM 161: 4 T 0.1mM 73A 4 TETRA TETRA 1.0mM 92 ± 3 FIG. 4. Effect of 0.5 mM tetracaine (TETRA) on secretion stimulated by 0.1 mM ACh or by excess potassium (65 mM). Maxi- * Maximum secretion = 82 nmol/mg of protein = 4.5 14 2.7 (SD) X mum secretion = 48 nmol/mg of protein = 6.3 ± 3.4 (SD) X con- control. trol. Downloaded by guest on September 27, 2021 Cell Biology: Schneider et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5039

z 0 loo0 w c, w C/) 80F- z 0

x w 6C * (n

D ACh ACh ACh NICOT NICOT NICOT MUSC PILO 40 x + + HEXAMETH HEXAMETH (10-4) (5 X 10-4) (10-4) (5 X 10 4) 0'. 20 FIG. 5. Effects of nicotinic and muscarinic drugs on secretion. ACh = 0.1 mM; hexamethonium (HEXAMETH) 0.1 and 0.5 mM; nicotine (NICOT) = 10 ,M; muscarine (MUSC) = 1.0-0.01 mM; I pilocarpine (PILO) = 1.0-0.01 mM. Maximum secretion = 85 nmol/ ACh ACh VINBL ACh CYTO B mg of protein = 6.8 + 4.0 (SD) X control. + + VINBL CYTO B of vinblastine alone. Cytochalasin B (1.0-10 MM) gave a small FIG. 6. Effects of microtubule and microfilament disrupting but possibly insignificant enhancement of secretion in the agents on secretion. ACh = 0.1 mM; vinblastine (VINBL) = 50M4M; presence of 0.1 mM ACh. However, cytochalasin B alone also cytochalasin B (CYTO B) = 1.0-10,uM. Maximum secretion = 60 induced a small secretion (19%) which, if subtracted from its nmol/mg of protein = 9.6 ± 3.5 (SD) X control. effect in the presence of ACh, would leave no significant net effect. The cholinergic receptors were found to be predominantly nicotinic based on (i) the ability of nicotine to stimulate secre- DISCUSSION tion, (fi) the lack of substantial secretion induced by muscarinic Bovine adrenal chromaffin cells have been isolated in large agonists, and (imi) the block of secretion by hexamethonium. quantity, shown to be viable, and characterized in terms of their Atropine (not shown) was also found to block the ACh response. catecholamine secretory function. The isolated cells exhibited However, this may not be due to its action as a specific mus- a normal secretory response to ACh and demonstrated a key carinic antagonist because we have also found it to block ni- requirement for calcium as mediator between cell surface cotine stimulation of catecholamine secretion. Interestingly, stimulation and exocytotic secretion. The conditions of cate- Smith and Winkler (20) has also noted the lack of effect of cholamine release from the isolated bovine chromaffin cells muscarinic drugs on bovine adrenal medullary secretion, and were generally in agreement with earlier data on the perfused this may be a peculiarity of the species, because muscarinic adrenal gland (3, 4) and on mixed medullary and cortical agents are known to be active in other mammals (3). Wilson and adrenal cell suspensions isolated from the guinea pig (9). New Kirshner (21) have similarly confirmed the entirely nicotinic data are provided for isolated chromaffin cells on the differ- nature of ACh receptors in bovine adrenal medulla. ential release of epinephrine and norepinephrine relative to We observed a difference between ACh- and excess K+- their content in the cells and medulla. In addition, the effects stimulated secretion with regard to the amount of catechol- of microtubule and microfilament disrupting agents on cate- amine released, the calcium dependence of the release, and the cholamine secretion were determined, and a comparison of ability of tetracaine to block release. Differences between ACh- stimulation by ACh versus excess potassium was made. and K+-stimulated secretion (9, 22-24) and changes in the The secretory response to ACh was rapid, with detectable frequency of chromaffin cell action potentials (7) have been release of catecholamines occurring on a time scale of seconds previously noted. Brandt et al. (7) demonstrated that an in- and 85% of maximum release occurring in 5 min. The dose- creased action potential spike frequency (2.5/sec) could be response curve of ACh-stimulated secretion had its half-max- maintained as long as 10,uM ACh was present in the medium, imum value at 10MM ACh and its maximum at 0.1 mM ACh. whereas 15.6 mM K+ was only able to increase the spike fre- These values are in agreement with ACh concentrations re- quency for about a minute, after which it dropped by an order quired to increase action potential spike frequencies in isolated of magnitude to spontaneous spike frequencies of about 0.1/sec. rat chromaffin cells (7) and to induce secretion from perfused If the cellular Ca2+ entry required for secretion occurred during glands (1, 19). Isolated guinea pig mixed adrenal cells have been each action potential, this might explain the greater catechol- reported to require an ACh concentration one order of mag- amine release by ACh than by K+ during prolonged incubations nitude higher for a similar secretory response (9). (20 min) and possibly the difference in Ca2+ dependence. Such The differential release of epinephrine and norepinephrine a mechanism might also explain the findings of Douglas and reflected their proportions in the chromaffin cells and suggest Rubin (22) that ACh will augment secretion from adrenal that saturating values of ACh (0.1 mM) are equally effective glands already depolarized with excess K. Alternatively, ACh in stimulating epinephrine- and norepinephrine-containing and K depolarization may affect different calcium permeability cells. It will be interesting in future studies to test various se- sites. Further work is needed to clarify this point. cretagogues and drugs for their ability to induce or inhibit either Our results with vinblastine would be consistent with, but do epinephrine or norepinephrine release selectively. This could not prove,-a requirement for intact microtubules in the secre- have implications for regulation of the physiological actions of tory mechanism. The question of cytoskeletal elements being catecholamines at a and 3 adrenergic receptors on many target involved in the translocation of chromaffin granules and exo- organs. cytosis is controversial. Poisner and Bernstein (18) have shown Downloaded by guest on September 27, 2021 5040 Cell Biology: Schneider et al. Proc. Nati. Acad. Sci. USA 74 (1977) that antimicrotubule agents inhibit catecholamine secretion, 1. Douglas, W. W. & Rubin, R. P. (1961) J. Physlol. (London) 159, whereas 2H20, a microtubule stabilizer, enhances secretion. 40-57. Poisner and Cooke (17) have performed competitive receptor 2. Douglas, W. W. (1968) Br. J. Pharmacol. 34,451-474. blocking studies and claim that neither vinblastine nor colchi- 3. Douglas, W. W. (1975) in HandbookofPhysiology, eds. Blaschko, cine acts at nicotinic receptors (17). Instead, they find vin- H., Sayers, G. & Smith, A. D. (American Physiology Society, Washington, DC), Sec. 7, Vol. 6, pp. 367-388. blastine competition with calcium, which could be at a tubulin 4. Viveros, 0. H. (1975) in Handbook ofPhysiology, eds. Blaschko, binding site. On the other hand, both Trifaro et al. (23) and H., Sayers, G. & Smith, A. D. (American Physiology Society, Douglas and Sorimachi (24) have noted that, whereas colchicine Washington, DC), Sect. 7, Vol. 6, pp. 389-426. and vinblastine inhibited ACh-induced secretion, these agents 5. Douglas, W. W., Kanno, T. & Sampson, S. R. (1967) J. Phystol. did not affect K+-stimulated secretion. If the site of action were (London) 188, 107-120. microtubules, one might expect an inhibition of K+-stimulated 6. Douglas, W. W., Kanno, T. & Sampson, S. R. (1967) J. Phystol. release because both K+ and ACh are thought to induce se- (London) 191, 107-121. cretion by a common pathway involving exocytosis. It is in- 7. Brandt, B. L., Hagiwara, Y., Kidokoro, Y. & Miyazaki, S. (1976) teresting that vinblastine has different effects on ACh- and J. Physiol. (London) 263,417-439. 8. Biales, B., Dichter, M. & Tischler, A. (1976) J. Physiol. (London) K+-stimulated release (23, 24), and this may be related to the 262,743-753. parallel differences we observed between ACh and excess K+ 9. Hochman, J. & Perlman, R. L. (1976) Biochim. Biophys. Acta with regard to calcium dependence and tetracaine block of 421, 168-175. secretion. (See note added in proof.) 10. Kloppenberg, P. W. C., Island, D. P., Little, G. W., Michelakis, From the previous literature on chromaffin cells and the data A. M. & Nicholson, W. E. (1968) Endocrinology 82, 1053- reported here, there is good reason to consider this system to be 1058. an excellent model for elucidating the mechanisms of both 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. hormone and neurotransmitter secretion. Furthermore, the (1951) J. Btol Chem. 193, 265-275. isolated cell system may be a useful system for studying the 12. von Euler, U. S. & Floding, L. (1955) Acta Physiol. Scand. Suppl. mechanism of action of hormones and 118, 33,45-56. neurotransmitters at their 13. von Euler, U. S. & Lishajko, F. (1961) Acta Physiol. Scand. 51, cell surface receptors. Indeed, Sonenberg and Schneider (25) 348-356. have recently proposed a model of hormone action that draws 14. Anton, A. H. & Sayre, D. F. (1962) J. Pharmacol. Exp. Ther. 138, heavily on analogy to neurotransmitter mechanisms and in- 360-375. cludes both an anionic and a cyclic nucleotide response. For the 15. von Euler, U. S. & Hamberg, U. (1950) Acta Phystol. Scand. 19, chromaffin cell and ACh receptor system there already exists 74-84. an important body of information on membrane electric po- 16. Shepherd, D. M. & West, G. B. (1953) J. Phystol. (London) 120, tential and ion permeability changes (5-8). There are also recent 15-19. data on the adrenal medulla and related systems, including 17. Poisner, A. M. & Cooke, P. (1975) Ann. N.Y. Acad. Sci. 253, postganglionic sympathetic neurons, demonstrating an eleva- 653-669. 18. Poisner, A. M. & Bernstein, J. (1971) J. Pharmacol. Exp. Ther. tion in cyclic nucleotides in response to ACh (26, 27). Thus, 177,102-108. there is the possibility that both a cyclic nucleotide and an ionic 19. Kimura, K. (1974) Sct. Rep. Nayora Women's Junior Coll. 7, response may occur via ACh receptors on the chromaffin cell 11-18 (cited in ref. 7). membrane. This would provide an excellent opportunity for 20. Smith, A. D. & Winkler, H. (1972) in Handbuch der Experi- elucidating their integrated role in regulating cellular metab- mentellen Pharmakologle, eds. Blaschko, H. & Muscholl, E. olism and secretion. (Springer, Berlin), Vol. 33, p. 584. 21. Wilson, S. P. & Kirshner, N. (1977) J. Neurochem. 28, 687- 695. Note Added in Proof. We have recently found that, in contrast to its 22. Douglas, W. W. & Rubin, R. P. (1963) J. Phystol. (London) 167, block of ACh-stimulated secretion, vinblastine has no significant effect 288-310. on K+-stimulated secretion from the isolated chromaffin cells. 23. Trifaro, J. M., Coller, B., Lastowecka, A. & Stern, D. (1972) Mol. Pharmacol. 8, 264-267. 24. Douglas, W. W. & Sorimachi, M. (1972) Br. J. Pharmacol. 45, It is a pleasure to thank Prof. Martin Sonenberg for introducing us 129-132. to the problem of cell surface regulation and for many stimulating and 25. Sonenberg, M. & Schneider, A. S. (1977) in Receptors and Rec- useful discussions. K.R. thanks Prof. Sonenberg for his hospitality at ognition, eds. Cuatrecasas, P. & Greaves, M. F. (Chapmann and Sloan-Kettering Institute. This work was supported in part by National Hall, London), Series A, Vol. 4, pp. 1-73. Science Foundation Grant PCM 76-04079; by National Institutes of 26. Guidotti, A. & Costa, E. (1977) Biochem. Pharmacol. 26,817- Health Grants CA 08748, CA 18759, and CA 15773; and by a grant 823. from the U.S.-Israel Binational Science Foundation (BSF), Jerusalem, 27. Kebabian, J. W., Bloom, F. E., Steiner, A. L. & Greengard, P. Israel. (1975) Science 190, 157-159. Downloaded by guest on September 27, 2021