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Exocytosis from a single rat chromaffin cell by cholinergic and peptidergic neurotransmitters
Pergamon
03064522(93)EOO58-X
NeuroscienceVol. 59, No. I, pp. l-5, 1994 ElsevierScienceLtd Copyright 0 1994 IBRO Printedin Great Britain. All rights reserved 0306-4522194$6.00 + 0.00
Letter to neuroscience EXOCYTOSIS FROM A SINGLE RAT CHROMAFFIN BY CHOLINERGIC AND PEPTIDERGIC NEUROTRANSMITTERS
CELL
P. S. CHOWDHURY, X. Guo, T. D. WAKADE,* D. A. PRZYWARA and A. R. WAKADE Department of Pharmacology, Wayne State University, School of Medicine, 540 East Canfield, Detroit, MI 48201. U.S.A.
Secretion of catecholamines from chromaffin ceils is mediated by cholinergic and peptidergic neurotransmitters.3,“*‘4 The choline@ transmitter acetylcholine activates both nicotinic and muscarinic receptors to trigger catecholamine secretion in rat adrenal medulla.” Vasoactive intestinal polypeptide (VIP) has been identified as the peptidergic transmitter in rat adrenal medulla and may also be the non-cholinergic transmitter in bovine adrenal.18su Pituitary adenylate cyclase activating polypeptide (PACAP), a VIP-like secretin peptide, is also found in the adrenal,’ and is a potent secretagogue. ‘J’ Thus, PACAP may be another peptidergic transmitter at the adrenal synapse.19 A most intriguing property of rat chromaffin cells is that stimulation of nicotinic, muscarinic, VIP or PACAP receptors are each able to produce robust catecholamine secretion on their own.‘1-13,21This raises the question of whether a single chromaibn cell can respond to each of the above agonists or whether the secretion is due to subpopulations of chromaiIin cells. This issue was addressed by using electrochemical techniques to monitor exocytosis from individual chromaffin cells in culture.2~9~22 We demonstrate that acetylcholine, nicotine, muscarine, VIP and PACAP are each able to evoke catecholamine secretion from a single chromaffin cell. Some cells only responded to acetylcholine. Furthermore, each agonist produced a distinct pattern of exocytosis. Muscarineevoked secretion exhibited a latency of OS-2 s, but exocytosis persisted up to 30 s following 500 ms stimulation. Nicotine produced an immediate response which usually ended within 10 s. The secretory pattern following acetylcholine appeared to be the sum of the nicotinic and muscarinic patterns, showing both rapid onset and longer duration. The unique property of peptidergic stimulation was that a brief exposure caused exocytosis to persist for up to 2min. Like muscarine, peptides exhibited latency in *To whom correspondence should be addressed. Abbreviations: PACAP, pituitary adenylate cyclase activating polypeptide; VIP, vasoactive intestinal polypeptide.
producing exocytosis. These findings support the idea that the interactive control of catecholamine secretion by nicotinic, muscarinic and peptidergic receptors occurs at the level of single cells. Exocytotic events recorded from a single rat chromaffin cell following 500 ms pressure ejection of five different agonists from a microcapillary on to the cell are shown in Fig. 1. Acetylcholine (10 p M) produced an immediate secretory response which lasted more than 20 s. Nicotine (10 PM) also produced an immediate response, but secretion stopped between 5 and 10 s after stimulation. Muscarine (30 PM), on the other hand, evoked a long-lasting secretion (approximately 20 s) but only after a 2-s latency. The peptides PACAP (0.1 p M) and VIP (20 p M) stimulated exocytosis from the cell as did cholinergic agonists (Fig. 1). Cells which responded to cholinergic agonists did not all respond to peptidergic agonists. Of 32 cells tested with acetylcholine and PACAP, 21 (66%) responded to both agonists. The average secretory pattern from eight chromaffin cells exposed to each of the five agonists is shown in Fig. 2. The exocytotic events are combined in successive two minute periods following a single 500 ms application of the agonist. Four response patterns are apparent: (i) acetylcholine produced the greatest number of exocytotic events throughout the recording period; (ii) nicotine produced a response similar to acetylcholine initially, but it then declined rapidly; (iii) only muscarine showed significantly less exocytosis in the first 2-min period; and (iv) VIP and PACAP produced significantly fewer exocytotic events than the cholinergic agonists. This general pattern of exocytosis was observed regardless of the order of agonist exposure in cells treated with all agonists, and also in cells stimulated with only a single agonist (not shown). The latency following muscarine exposure ranged from 0.5 to 2 s in 16 cells tested. Exocytotic events following 500 ms exposure to PACAP also exhibited latency and were observed for up to 2min after stimulation (not shown).
P. S, CH~WUHURY
Musyarine
1
1
_,
_
et uf.
~
i
__
0
PACAP l
VIP h
-
h&
l
Fig. 1.Exocytosis measured from a single chromaffin cell stimulated by five different agonists. Traces show current resulting from the oxidation of catecholamines at the exposed tip of an insulated &pm diameter carbon fiber placed within 1 pm of the cell (holding potential 650 mV). One second after the start of recording, agonist dissolved in bath solution was applied for 500ms (dot) by pressure ejection from a micro capillary aimed at the cell. Agonist concentrations were: acetylcholine, 10PM; nicotine, 10FM; muscarine, 30 pM; PACAP, 0.1 PM; and VIP, 2OpM. Traces shown are 15 s. ~l~tr~herni~l techniques were as described by Leszczyszyn et al.,9 using the amperometric mode of an Ensman Potentiostat coupled to a personal computer. Carbon fiber electrodes were made as described by Chow er at.* Chromaffin cells were isolated from two-to four-week-old rat pups by enzymatic digestion.‘“~16-Cells in M-199 medium with
15% fetal calf serum and 0.1 PM dexamethasone were plated on 35-mm collagen coated culture dishes and used after two days.
A common stimulation paradigm used to study secretion from chromaffin cells in the intact adrenal and in cultural is to apply agonist for several seconds to several minutes.1~g~‘5~20 We questioned whether the duration of stimulation would reveal further differences in exocytotic patterns evoked by the five agonists. Exocytosis was monitored during 2min of continuous exposure to each of the five agonists. Figure 3 shows that each agonist produced exocytosis throughout the stimulation period. Even nicotine, which was expected to cause rapid desensitization of the chromaffin cell acetylcholine receptor,4 continued to evoke a large number of exocytotic events during 2 min stimulation. Earlier work with perfused adrenal gland could not rule out the possible involvement of an intermediate
cell type in the stimulatory action of VIP. Here we clearly show that VIP acts directly on chromaffin cells to evoke catecholamine secretion. The same was true for muscarinic stimulation. Activation of muscarinic receptors is not coupled to secretion in bovine adrenal chromaffin cells in culture and has been regarded as physiologically unimportant. Unfortunately, there is no evidence for or against a muscarinic component of secretion in the intact bovine adrenal. It may be that culture conditions alter muscarinic pathways in bovine chromafhn cells in culture. Our results clearly show that muscarinic receptors pIay an important role in secretion of catecholamines from rat chromaffin cells in culture and in the intact gland.‘2~“~‘7 The reason for the characteristic delay in exocytosis following muscarinic stimulation is not known. It will
Exocytosis from a single rat chromaffin cell 8 1I ~ n -
ACETYLCHOL[NE
~[t111=¢~m
6 4
2
0
be important to know whether a similar delay occurs in the muscarine-stimulated rise in intracellular C a 2+ concentration or whether some other component of the muscarine stimulated pathway accounts for the latency. Regardless of the mechanism involved, the data show that nicotinic, muscarinic and peptidergic receptors are all present and coupled to secretion in individual chromaffin cells. These results offer strong support for the idea that interactive control of catecholamine secretion by nicotinic, muscarinic and peptidergic receptors in the at a
ra s
to c o
ommnic
o
o
ent second messengers in individual chromaftin
NICOTINE
cells?3
6
The finding that secretion does not desensitize during minutes of continuous exposure to any ago-
4
1
O
.
~
.
I
~
.
2
.
861
.
1
.
~
.
~
.
.
MUSCARINE
0 0 x W
3
°
6
PACAP
4
8"
62
nist, including nicotine, is consistent with previous studies of secretion from chromaffin cells in the adrenal gland and in culture. In cultured chromaffin cells, desensitization of nicotine-evoked secretion was reported to be in the minute time range, ~while a 50% decrease in secretion from perfused adrenal required 10 min of continuous exposure to nicotine. ~2 Recent voltage-clamp data also show no decrease in receptor activated current during one minute continuous stimulation with nicotine or acetylcholine.8 These findings are consistent with the physiology of the adrenal during stress when continuous secretion of catecholamines is stimulated by acetylcholine acting on nicotinic and muscarinic receptors. We have also found that continuous exposure to nicotine (or muscarine) for 2 min causes a sustained elevation of intracellular Ca 2+ which does not desensitize (unpublished observations). The stimulation of catecholamine secretion from rat chromaffin cells through cholinergic and peptidergic transmitters provides a unique model to study how different transmitters through multiple receptors and possibly second messengers control exocytosis. This brief report demonstrates a great potential of cultured rat chromaffin cells to study exocytosis by physiological ligands and their interactions with second messengers and other components of the exocytotic machinery. A distinct advantage of the rat model is the wealth of information already available regarding the physiology of secretion in the perfused adrenal gland of the rat. The ability to compare secretory behavior of chromaffin cells in culture with the intact gland would allow identification of properties unique to culture which may be confounding our understanding of cellular mechanisms in exocytosis.
VIP 42
2
1'0 1'2 1'4 16 18 ELAPSED TIME (s) 4
6
8
Fig. 2. Time-course of exocytosis evoked by different agonists. Histograms show the number of exocytotic events (mean _+SEM, n - 8) in successive 2-s periods following 500 ms application of agonists. Each cell was stimulated with each agonist in varied order of application. Deflections less than 4 x the background noise level (i.e. < 2.0 pA) were not included. Other details as in Fig. 1.
P. S. CHOWDHURY et al
Fig. 3. Exocytosis during 2 mitt of continuous stimulation. Agonist application was started 15 s after beginning recordings (arrow) and continued throughout the 2&n recording period. Acetylcholine and nicotine response from the same ceil, muscarine and VIP from a different ceil and PACAP response from a third cell. Am~romet~c recordings as in Fig. 1.
REFERENCES i.
2. 3. 4. 5. 6. 7.
Boksa P. and Livett B. G. (1984) Desensitization to nicotinic choiinergic agonists and K+, agents that stimulate catechoiamine secretion, in isolated adrenal chromaffin ceils. J. Neurochem. 42, 6077617. Chow R. H., von Ruden L. and Neher E. (1992) Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin ceils. Nuture 356, 60-63. Edwards A. V. and Jones C. T. (1989) Adrenal responses to spianchnic nerve stimulation in conscious calves given naloxone. J. Physiol. 418, 339-351. Fenwick E. M., Marty A. and Neher E. (1982) A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetyichoiine. J. Physioi. 331, 577-597. Gottschaii P. E., Tatsuno I., Miyata A. and Arimura A. (1990) Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyciase-activating poiypeptide. Endocrinology 127, 272-277. Guo X. and Wakade A. R. (1994) Differential secretion of catechoiamines in response to peptidergic and choiinergic transmitters in the rat adrenal. J. Physiot. (in press). Isobe K., Nakai T. and Takawa Y. (1993) Ca**-dependent stimuiatory affect of pituitary adenyiate cyciase activating poiypeptide on catechoiamine secretion from cultured porcine adrenal meduiiary chromaffin ceils. Endocrinology 132,
1757-1765. 8. Kiepper M., Hans M. and Takeda K. (1990) Nicotinic choiinergic modulation of voltage-dependent calcium current in bovine adrenal chromaffin ceils. J. Physiol. 428, 545-560. 9. Leszczyszyn D. J., Jankowski J. A., Viveros 0. H., Diiiberto E. J., Near D. J. and Whightman R. M. (1990)
Nieotinic-receptor mediated catecholamine secretion From individual chromaffin ceils: chemical evidence for exocytosis. J. biol. Chem. 265, 14,73614,737.
10. Liiiien L. E. and Claude P. (1985) Nerve growth factor is a mitogen for cultured chromaffin cells. Nature 317,6233634. 1 I, Maihotra R. K. and Wakade A. R. (1987) Vasoactive intestinal poiypeptide stimulates the secretion of catechoiamines from the rat adrenal gland. J. Physiof, 388, 28$_294. 12. Malhotra R. K., Wakade T. D. and Wakade A. R. (1988) Comparison of secretion of catecholamiaes from the rat adrenal medulla during continuous exposure to nicotine, muscarine or excess K. Neuroscience 26, 313-320. 13. Maihotra R. K., Wakade T. D. and Wakade A. R. (1989) Cross-communication between acetyichoiine and VIP in controlling catechoiamine secretion by affecting CAMP, inositoi triphosphate, protein kinase C, and calcium in rat adrenal medulla. J. Neurosci. 9, 4150-4157.
Exocytosis
from a single rat chromaffin
cell
5
14. Marley P. D., Thomson K. A. and Smardencas A. (1993) Non-cholinergic nervous control of catecholamine secretion from perfused bovine adrenal glands. J. Physiol. 465, 489-500. 15. Uceda G., Artalejo A. R., Lopez M. G., Abad F., Neher E. and Garcia A. G. (1992) Ca’+-activated K+ channels modulate muscarinic secretion in cat chromaffin cells. J. Physiol. 454, 213-230. 16. Unsicker K., Krisch B., Otten U. and Theonen H. (1978) Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin by glucocorticoids. Proc. natn. Acad. Sci. U.S.A. 75, 3498-3502. 17. Wakade A. R. and Wakade T. D. (1983) Contribution of nicotinic and muscarinic receptors in the secretion of catecholamines evoked by endogenous and exogenous acetylcholine. Neuroscience 10, 973-978. 18. Wakade T. D., Blank M. A., Malhotra R. K., Pourcho R. and Wakade A. R. (1991) The peptide VIP is a neurotransmitter in rat adrenal medulla: physiological role in controlling catecholamine secretion. J. Physiol. 444, 349-362. 19. Wakade A. R., Guo X., Strong R., Arimura A. and Haycock J. (1992) Pituitary adenylate cyclase-activating polypeptide (PACAP) as a neurotransmitter in rat adrenal medulla. Regul. Pept. 37, 331. 20. Warashina A., Fujiwara N. Hirano T. and Shimoji K. (1988) Characteristics of bradykinin-evoked secretory response in the perfused rat adrenal medulla. Biomed. Res. 9, 139-145. 21. Watanabe T., Masuo Y., Matsumoto H., Suzuki N., Ohtaki T., Masuda Y., Kitada C., Tsuda M. and Fujino M. (1992) Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline. Biochem. biophys. Res Commun. 182, 403411. 22. Whightman R. M., Jankowski J. A., Kennedy R. T., Kawagoe K. T., Schroeder T. J., Leszczyszyn D. J., Near J. A., Diliberto E. J., Viveros 0. H. (1991) Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. natn. Acad. Sci. U.S.A. 88, 10,75410,758. 23. Yoshikawa M., Saito H., Sano T., Ohuchi T., Ishimura Y., Morita K., Saito S. and Oka M. (1990) Localization and release of immunoreactive vasoactive intestinal polypeptide in bovine adrenal medulla Neurosci. Lett. 111, 75-79. (Accepted 22 November 1993)
03064522(93)EOO58-X
NeuroscienceVol. 59, No. I, pp. l-5, 1994 ElsevierScienceLtd Copyright 0 1994 IBRO Printedin Great Britain. All rights reserved 0306-4522194$6.00 + 0.00
Letter to neuroscience EXOCYTOSIS FROM A SINGLE RAT CHROMAFFIN BY CHOLINERGIC AND PEPTIDERGIC NEUROTRANSMITTERS
CELL
P. S. CHOWDHURY, X. Guo, T. D. WAKADE,* D. A. PRZYWARA and A. R. WAKADE Department of Pharmacology, Wayne State University, School of Medicine, 540 East Canfield, Detroit, MI 48201. U.S.A.
Secretion of catecholamines from chromaffin ceils is mediated by cholinergic and peptidergic neurotransmitters.3,“*‘4 The choline@ transmitter acetylcholine activates both nicotinic and muscarinic receptors to trigger catecholamine secretion in rat adrenal medulla.” Vasoactive intestinal polypeptide (VIP) has been identified as the peptidergic transmitter in rat adrenal medulla and may also be the non-cholinergic transmitter in bovine adrenal.18su Pituitary adenylate cyclase activating polypeptide (PACAP), a VIP-like secretin peptide, is also found in the adrenal,’ and is a potent secretagogue. ‘J’ Thus, PACAP may be another peptidergic transmitter at the adrenal synapse.19 A most intriguing property of rat chromaffin cells is that stimulation of nicotinic, muscarinic, VIP or PACAP receptors are each able to produce robust catecholamine secretion on their own.‘1-13,21This raises the question of whether a single chromaibn cell can respond to each of the above agonists or whether the secretion is due to subpopulations of chromaiIin cells. This issue was addressed by using electrochemical techniques to monitor exocytosis from individual chromaffin cells in culture.2~9~22 We demonstrate that acetylcholine, nicotine, muscarine, VIP and PACAP are each able to evoke catecholamine secretion from a single chromaffin cell. Some cells only responded to acetylcholine. Furthermore, each agonist produced a distinct pattern of exocytosis. Muscarineevoked secretion exhibited a latency of OS-2 s, but exocytosis persisted up to 30 s following 500 ms stimulation. Nicotine produced an immediate response which usually ended within 10 s. The secretory pattern following acetylcholine appeared to be the sum of the nicotinic and muscarinic patterns, showing both rapid onset and longer duration. The unique property of peptidergic stimulation was that a brief exposure caused exocytosis to persist for up to 2min. Like muscarine, peptides exhibited latency in *To whom correspondence should be addressed. Abbreviations: PACAP, pituitary adenylate cyclase activating polypeptide; VIP, vasoactive intestinal polypeptide.
producing exocytosis. These findings support the idea that the interactive control of catecholamine secretion by nicotinic, muscarinic and peptidergic receptors occurs at the level of single cells. Exocytotic events recorded from a single rat chromaffin cell following 500 ms pressure ejection of five different agonists from a microcapillary on to the cell are shown in Fig. 1. Acetylcholine (10 p M) produced an immediate secretory response which lasted more than 20 s. Nicotine (10 PM) also produced an immediate response, but secretion stopped between 5 and 10 s after stimulation. Muscarine (30 PM), on the other hand, evoked a long-lasting secretion (approximately 20 s) but only after a 2-s latency. The peptides PACAP (0.1 p M) and VIP (20 p M) stimulated exocytosis from the cell as did cholinergic agonists (Fig. 1). Cells which responded to cholinergic agonists did not all respond to peptidergic agonists. Of 32 cells tested with acetylcholine and PACAP, 21 (66%) responded to both agonists. The average secretory pattern from eight chromaffin cells exposed to each of the five agonists is shown in Fig. 2. The exocytotic events are combined in successive two minute periods following a single 500 ms application of the agonist. Four response patterns are apparent: (i) acetylcholine produced the greatest number of exocytotic events throughout the recording period; (ii) nicotine produced a response similar to acetylcholine initially, but it then declined rapidly; (iii) only muscarine showed significantly less exocytosis in the first 2-min period; and (iv) VIP and PACAP produced significantly fewer exocytotic events than the cholinergic agonists. This general pattern of exocytosis was observed regardless of the order of agonist exposure in cells treated with all agonists, and also in cells stimulated with only a single agonist (not shown). The latency following muscarine exposure ranged from 0.5 to 2 s in 16 cells tested. Exocytotic events following 500 ms exposure to PACAP also exhibited latency and were observed for up to 2min after stimulation (not shown).
P. S, CH~WUHURY
Musyarine
1
1
_,
_
et uf.
~
i
__
0
PACAP l
VIP h
-
h&
l
Fig. 1.Exocytosis measured from a single chromaffin cell stimulated by five different agonists. Traces show current resulting from the oxidation of catecholamines at the exposed tip of an insulated &pm diameter carbon fiber placed within 1 pm of the cell (holding potential 650 mV). One second after the start of recording, agonist dissolved in bath solution was applied for 500ms (dot) by pressure ejection from a micro capillary aimed at the cell. Agonist concentrations were: acetylcholine, 10PM; nicotine, 10FM; muscarine, 30 pM; PACAP, 0.1 PM; and VIP, 2OpM. Traces shown are 15 s. ~l~tr~herni~l techniques were as described by Leszczyszyn et al.,9 using the amperometric mode of an Ensman Potentiostat coupled to a personal computer. Carbon fiber electrodes were made as described by Chow er at.* Chromaffin cells were isolated from two-to four-week-old rat pups by enzymatic digestion.‘“~16-Cells in M-199 medium with
15% fetal calf serum and 0.1 PM dexamethasone were plated on 35-mm collagen coated culture dishes and used after two days.
A common stimulation paradigm used to study secretion from chromaffin cells in the intact adrenal and in cultural is to apply agonist for several seconds to several minutes.1~g~‘5~20 We questioned whether the duration of stimulation would reveal further differences in exocytotic patterns evoked by the five agonists. Exocytosis was monitored during 2min of continuous exposure to each of the five agonists. Figure 3 shows that each agonist produced exocytosis throughout the stimulation period. Even nicotine, which was expected to cause rapid desensitization of the chromaffin cell acetylcholine receptor,4 continued to evoke a large number of exocytotic events during 2 min stimulation. Earlier work with perfused adrenal gland could not rule out the possible involvement of an intermediate
cell type in the stimulatory action of VIP. Here we clearly show that VIP acts directly on chromaffin cells to evoke catecholamine secretion. The same was true for muscarinic stimulation. Activation of muscarinic receptors is not coupled to secretion in bovine adrenal chromaffin cells in culture and has been regarded as physiologically unimportant. Unfortunately, there is no evidence for or against a muscarinic component of secretion in the intact bovine adrenal. It may be that culture conditions alter muscarinic pathways in bovine chromafhn cells in culture. Our results clearly show that muscarinic receptors pIay an important role in secretion of catecholamines from rat chromaffin cells in culture and in the intact gland.‘2~“~‘7 The reason for the characteristic delay in exocytosis following muscarinic stimulation is not known. It will
Exocytosis from a single rat chromaffin cell 8 1I ~ n -
ACETYLCHOL[NE
~[t111=¢~m
6 4
2
0
be important to know whether a similar delay occurs in the muscarine-stimulated rise in intracellular C a 2+ concentration or whether some other component of the muscarine stimulated pathway accounts for the latency. Regardless of the mechanism involved, the data show that nicotinic, muscarinic and peptidergic receptors are all present and coupled to secretion in individual chromaffin cells. These results offer strong support for the idea that interactive control of catecholamine secretion by nicotinic, muscarinic and peptidergic receptors in the at a
ra s
to c o
ommnic
o
o
ent second messengers in individual chromaftin
NICOTINE
cells?3
6
The finding that secretion does not desensitize during minutes of continuous exposure to any ago-
4
1
O
.
~
.
I
~
.
2
.
861
.
1
.
~
.
~
.
.
MUSCARINE
0 0 x W
3
°
6
PACAP
4
8"
62
nist, including nicotine, is consistent with previous studies of secretion from chromaffin cells in the adrenal gland and in culture. In cultured chromaffin cells, desensitization of nicotine-evoked secretion was reported to be in the minute time range, ~while a 50% decrease in secretion from perfused adrenal required 10 min of continuous exposure to nicotine. ~2 Recent voltage-clamp data also show no decrease in receptor activated current during one minute continuous stimulation with nicotine or acetylcholine.8 These findings are consistent with the physiology of the adrenal during stress when continuous secretion of catecholamines is stimulated by acetylcholine acting on nicotinic and muscarinic receptors. We have also found that continuous exposure to nicotine (or muscarine) for 2 min causes a sustained elevation of intracellular Ca 2+ which does not desensitize (unpublished observations). The stimulation of catecholamine secretion from rat chromaffin cells through cholinergic and peptidergic transmitters provides a unique model to study how different transmitters through multiple receptors and possibly second messengers control exocytosis. This brief report demonstrates a great potential of cultured rat chromaffin cells to study exocytosis by physiological ligands and their interactions with second messengers and other components of the exocytotic machinery. A distinct advantage of the rat model is the wealth of information already available regarding the physiology of secretion in the perfused adrenal gland of the rat. The ability to compare secretory behavior of chromaffin cells in culture with the intact gland would allow identification of properties unique to culture which may be confounding our understanding of cellular mechanisms in exocytosis.
VIP 42
2
1'0 1'2 1'4 16 18 ELAPSED TIME (s) 4
6
8
Fig. 2. Time-course of exocytosis evoked by different agonists. Histograms show the number of exocytotic events (mean _+SEM, n - 8) in successive 2-s periods following 500 ms application of agonists. Each cell was stimulated with each agonist in varied order of application. Deflections less than 4 x the background noise level (i.e. < 2.0 pA) were not included. Other details as in Fig. 1.
P. S. CHOWDHURY et al
Fig. 3. Exocytosis during 2 mitt of continuous stimulation. Agonist application was started 15 s after beginning recordings (arrow) and continued throughout the 2&n recording period. Acetylcholine and nicotine response from the same ceil, muscarine and VIP from a different ceil and PACAP response from a third cell. Am~romet~c recordings as in Fig. 1.
REFERENCES i.
2. 3. 4. 5. 6. 7.
Boksa P. and Livett B. G. (1984) Desensitization to nicotinic choiinergic agonists and K+, agents that stimulate catechoiamine secretion, in isolated adrenal chromaffin ceils. J. Neurochem. 42, 6077617. Chow R. H., von Ruden L. and Neher E. (1992) Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin ceils. Nuture 356, 60-63. Edwards A. V. and Jones C. T. (1989) Adrenal responses to spianchnic nerve stimulation in conscious calves given naloxone. J. Physiol. 418, 339-351. Fenwick E. M., Marty A. and Neher E. (1982) A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetyichoiine. J. Physioi. 331, 577-597. Gottschaii P. E., Tatsuno I., Miyata A. and Arimura A. (1990) Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyciase-activating poiypeptide. Endocrinology 127, 272-277. Guo X. and Wakade A. R. (1994) Differential secretion of catechoiamines in response to peptidergic and choiinergic transmitters in the rat adrenal. J. Physiot. (in press). Isobe K., Nakai T. and Takawa Y. (1993) Ca**-dependent stimuiatory affect of pituitary adenyiate cyciase activating poiypeptide on catechoiamine secretion from cultured porcine adrenal meduiiary chromaffin ceils. Endocrinology 132,
1757-1765. 8. Kiepper M., Hans M. and Takeda K. (1990) Nicotinic choiinergic modulation of voltage-dependent calcium current in bovine adrenal chromaffin ceils. J. Physiol. 428, 545-560. 9. Leszczyszyn D. J., Jankowski J. A., Viveros 0. H., Diiiberto E. J., Near D. J. and Whightman R. M. (1990)
Nieotinic-receptor mediated catecholamine secretion From individual chromaffin ceils: chemical evidence for exocytosis. J. biol. Chem. 265, 14,73614,737.
10. Liiiien L. E. and Claude P. (1985) Nerve growth factor is a mitogen for cultured chromaffin cells. Nature 317,6233634. 1 I, Maihotra R. K. and Wakade A. R. (1987) Vasoactive intestinal poiypeptide stimulates the secretion of catechoiamines from the rat adrenal gland. J. Physiof, 388, 28$_294. 12. Malhotra R. K., Wakade T. D. and Wakade A. R. (1988) Comparison of secretion of catecholamiaes from the rat adrenal medulla during continuous exposure to nicotine, muscarine or excess K. Neuroscience 26, 313-320. 13. Maihotra R. K., Wakade T. D. and Wakade A. R. (1989) Cross-communication between acetyichoiine and VIP in controlling catechoiamine secretion by affecting CAMP, inositoi triphosphate, protein kinase C, and calcium in rat adrenal medulla. J. Neurosci. 9, 4150-4157.
Exocytosis
from a single rat chromaffin
cell
5
14. Marley P. D., Thomson K. A. and Smardencas A. (1993) Non-cholinergic nervous control of catecholamine secretion from perfused bovine adrenal glands. J. Physiol. 465, 489-500. 15. Uceda G., Artalejo A. R., Lopez M. G., Abad F., Neher E. and Garcia A. G. (1992) Ca’+-activated K+ channels modulate muscarinic secretion in cat chromaffin cells. J. Physiol. 454, 213-230. 16. Unsicker K., Krisch B., Otten U. and Theonen H. (1978) Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin by glucocorticoids. Proc. natn. Acad. Sci. U.S.A. 75, 3498-3502. 17. Wakade A. R. and Wakade T. D. (1983) Contribution of nicotinic and muscarinic receptors in the secretion of catecholamines evoked by endogenous and exogenous acetylcholine. Neuroscience 10, 973-978. 18. Wakade T. D., Blank M. A., Malhotra R. K., Pourcho R. and Wakade A. R. (1991) The peptide VIP is a neurotransmitter in rat adrenal medulla: physiological role in controlling catecholamine secretion. J. Physiol. 444, 349-362. 19. Wakade A. R., Guo X., Strong R., Arimura A. and Haycock J. (1992) Pituitary adenylate cyclase-activating polypeptide (PACAP) as a neurotransmitter in rat adrenal medulla. Regul. Pept. 37, 331. 20. Warashina A., Fujiwara N. Hirano T. and Shimoji K. (1988) Characteristics of bradykinin-evoked secretory response in the perfused rat adrenal medulla. Biomed. Res. 9, 139-145. 21. Watanabe T., Masuo Y., Matsumoto H., Suzuki N., Ohtaki T., Masuda Y., Kitada C., Tsuda M. and Fujino M. (1992) Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline. Biochem. biophys. Res Commun. 182, 403411. 22. Whightman R. M., Jankowski J. A., Kennedy R. T., Kawagoe K. T., Schroeder T. J., Leszczyszyn D. J., Near J. A., Diliberto E. J., Viveros 0. H. (1991) Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. natn. Acad. Sci. U.S.A. 88, 10,75410,758. 23. Yoshikawa M., Saito H., Sano T., Ohuchi T., Ishimura Y., Morita K., Saito S. and Oka M. (1990) Localization and release of immunoreactive vasoactive intestinal polypeptide in bovine adrenal medulla Neurosci. Lett. 111, 75-79. (Accepted 22 November 1993)