- Email: [email protected]
Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells
Neuroscience Letters, 72 (1986) 315 319
315
Elsevier Scientific Publishers Ireland Ltd.
NSL 04325
Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells Mir Misbahuddin, Hitoshi Houchi, Atsushi Nakanishi, Kyoji Morita and Motoo O k a Department ofl Pharmacology, Tokushima University School of Medicine Tokushima (Japan) (Received 16 July 1986; Revised version received 2 September 1986; Accepted 3 September 1986)
Key wordsv Vasoactive intestinal polypeptide (VIP); Catecholamine secretion; Muscarinic receptor: Adrenal medullary cell; Guinea pig The effects of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion by isolated guinea pig adrenal medullary cells were studied. VIP (1 pM) alone induced only a slight secretion of CA, but it stimulated ACh-induced CA secretion. At concentrations of 0.01 l ,uM, it stimulated muscarine-induced CA secretion, but not nicotine-induced CA secretion. It did not affect high K + or Ca 2+ ionophore-induced CA secretion. The stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2 200/iM and was detectable after 2 min incubation.
The adrenal medulla is innervated by preganglionic sympathetic fibers from splanchnic nerves. Stimulation of these nerves causes release of acetylcholine (ACh) from the nerve terminals, which initiates the secretion of catecholamine (CA) from adrenal medullary cells. Recently, immunohistochemical studies demonstrated the presence of vasoactive intestinal polypeptide (VIP) not only in the splanchnic nerve terminals, but also in the adrenal medullary cells [2, 3, 10, 14]. These findings suggest that VIP is secreted with ACh from splanchnic nerve terminals or with CA from adrenal medullary cells, and modulates the secretion of CA mediated by ACh receptors. Therefore, we examined the effect of VIP on CA secretion from isolated guinea pig adrenal medullary cells. Results showed that VIP alone induced only a very slight CA secretion, but it significantly stimulated the secretion of CA induced by stimulation of muscarinic ACh receptors, but not that induced by stimulation of nicotinic ACh receptors. Guinea pigs (Hartley strain) of both sexes weighing 400 500 g were killed by a Correspondence. M. Oka, Department of Pharmacology, Tokushima University School of Medicine, 3 Kuramoto, T o k u s h i m a 770, Japan. 0304-3940,'86,,'$ 03.50 O 1986 Elsevier Scientific Publishers Ireland Ltd.
316
blow on the head and decapitation. In each experiment, 8-12 guinea pigs were killed and their adrenal glands were promptly removed. The medulla was separated from the adhering fat and the cortex with a pair of fine scissors, cut into several pieces and digested with collagenase as described elsewhere [13]. The isolated cells were suspended in Krebs-Ringer phosphate (KRP) buffer solution (in mM: NaC1 154, KCI 5.6, CaCI2 2.2, MgSO4 1.1, NaH2PO4 0.85, Na2HPO4 2.15, glucose 10 and 0.5% bovine serum albumin, pH 7.4) at a density of 5 to 7 x 105 cells/ml. The viability of the cells, determined by the Trypan blue exclusion test, was more than 95%. Bovine adrenal medullary cells, isolated by essentially the same method, were used in some experiments. The amount of CA released from the isolated adrenal medullary cells was measured by a modification of the trihydroxyindole method [9]. VIP was obtained from the Peptide Institute, Osaka, Japan. A peptidase inhibitor was not used. Table I shows the effects of VIP on basal CA secretion and CA secretion induced by the physiological secretagogue ACh, the nicotinic agonist nicotine and the muscarinic agonist muscarine. VIP alone at a concentration of I/zM induced only a slight secretion of CA, but it significantly stimulated ACh-induced CA secretion. It also stimulated muscarine-induced CA secretion dose-dependently, but did not stimulate nicotine-induced CA secretion. Its stimulatory effect on muscarine-induced CA secre-
TABLE I EFFECTS OF VIP ON BASAL AND INDUCED MEDULLARY CELLS
CA SECRETION FROM ISOLATED ADRENAL
The cells were preincubated (5 min, at 37°C) with or without VIP and then stimulated with ACh, muscarine, nicotine or high K + for 10 min or with A23187 for 30 min. After incubation, the cells were precipitated by centrifugation at 500 g for 5 min. CA in the cells and medium was extracted with 10% acetic acid and measured fluorometrically [9]. C A secretion was shown as a percentage of the total cellular C A content. The maximum secretion induced by each stimulant was expressed as 100%. Values are means __+S.E.M. of 3 ~ experiments. The effect of VIP was significant ( * P < 0 . 0 1 ) . Stimulant (/tM)
VIP (/zM)
CA secretion (% of total)
Stimulation (%)
-
1.0
--
ACh (200) ACh (200) Muscarine Muscarine Muscarine Muscarine
1.0 -0.01 0.1 1.0
1.3+0.2 2.5+0.4 7.8+0.3 11.3+0.4 6.3 + 0.4 7.7 + 0.5 8.5 + 0.5 9.2 _+0.6 4.6 _+0.3 5.7 + 0.6 6.0+0.5 5.9+0.5 9.6-t-0.8 10.1 + 0 . 8
(200) (200) (200) (200) N i c o t i n e (100) Nicotine (100) High K + (56 m M ) High K + (56 m M ) A23187 (10) A23187 (10)
1.0 1.0 1.0
100 145" 100 122 135* 146* 100 124
100 98
100 105
317
tion was detectable at 0.01 /tM and was significant at 0.1 and 1 /tM, the maximum concentration tested. We also examined the effect of VIP on CA secretion from isolated bovine adrenal medullary cells, from which secretion of CA is mainly induced by nicotinic stimulation. Results showed that VIP did not affect nicotine-induced CA secretion from isolated bovine adrenal medullary cells (data not shown). VIP also did not affect CA secretion caused by high K + and the Ca 2+ ionophore A23187 (Table 1). These results indicated that VIP has a selective stimulatory effect on muscarinic receptor-mediated CA secretion from isolated guinea pig adrenal medullary cells. As shown in Table II, the stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2-200/zM, with the highest concentration inducing the maximum response by itself. Fig. 1 shows the time course of stimulation by VIP of muscarine-induced CA secretion from isolated guinea pig adrenal medullary cells. Stimulation by VIP was detectable after 2 rain and persisted for at least 10 rain. It is interesting that VIP has a selective stimulatory effect on the muscarinic response. These results are consistent with recent electrophysiological findings that VIP potentiates muscarinic receptor-induced depolarization, but not nicotinic receptorinduced depolarization in superior cervical ganglionic cells [8]. The mechanism of the selective stimulatory effect of VIP on muscarinic responses is unknown. Secretion of CA by muscarinic stimulation from isolated guinea pig adrenal medullary cells was dependent on the presence of Ca 2+ in the medium, like the secretion of CA by nicotinic stimulation, suggesting that an increase in Ca 2+ uptake by the cells may be involved in the stimulation of CA secretion caused by muscarine. On the other hand, muscarinic stimulation is known to increase the cytoplasmic level of free Ca 2 +, which may be released from store sites [7, 12], increase phosphatidyl inositol turnover (the so-called PI response) [1, 4, 5, 17] and increase the cyclic G M P level in the cells [15, 18]. VIP may stimulate these responses and thereby muscarinic receptor-mediated CA secretion from the cells. In studies on a membrane preparation from the cat salivT A B L E I1 E F F E C T S O F VIP O N C A S E C R E T I O N I N D U C E D BY D I F F E R E N T
FROM
CONCENTRATIONS
ISOLATED ADRENAL
MEDULLARY
CELLS
OF MUSCARINE
The cells were i n c u b a t e d as described in T a b l e 1. C A secretion was s h o w n as a p e r c e n t a g e of the total C A in the cells. C A secretion induced by m u s c a r i n e w i t h o u t VIP was expressed as 100%. Values are the m e a n s o f 3 experiments. Muscarine (llM)
VIP (/~M)
C A secretion (% o f total)
Stimulation (%)
2 2 20 20 200 200
-1 -1
0.9 1.5 3.3 4.8 5.0 7.8
t00 166 100 149 100 156
I
318
10tO x-
O A O
.E ~E
5-
o
r, c,
£b 05
0 Time
10
(rain)
Fig. 1. Time course of stimulatory effect of VIP on muscarine-induced CA secretion from isolated adrenal medullary cells. Cells were incubated for various periods with musearine (200 #M) (O) or with muscarine (200/~M) plus VIP (1 ~tM) (0). CA secretion was shown as a percentage of the total cellular CA content. Points are means of two separate experiments.
ary gland, VIP was found to increase the binding of the muscarinic agonist [3H]Nmethyl-4-piperidinyl benzilate to membrane receptors [11]. Recently, Ip et al. [6] and Tischler et al. [16] found that VIP stimulated the synthesis of CA from tyrosine, by activation of tyrosine hydroxylase in rat sympathetic ganglion cells and rat PC 12 cells. In rat PC 12 cells, VIP increased the level of cyclic AMP [16], which activates tyrosine hydroxylase. In this experiment, the effect of VIP on the cyclic AMP level in isolated guinea pig adrenal medullary cells was not examined. However, since VIP stimulated muscarine-induced CA secretion from the cells whereas dibutyryl cyclic AMP did not (data not shown), it does not seem to stimulate muscarine-induced CA secretion by a mechanism involving cyclic AMP. Further studies are required on the mechanism of the effect of VIP in stimulating the muscarinic response.
We thank Mrs. Keiko Tachibana for typing this manuscript. This work was supported by grants from the Japanese Ministry of Education, Science and Culture. 1 Azila, N. and Hawthorne, J.N., Subcellular localization of phospholipid changes in response to muscarinic stimulation of perfused bovine adrenal medulla, Biochem. J., 204 (1982) 291--299. 2 Carmichael, S.W., The Adrenal Medulla, Vol. 3, Eden Press, Quebec, 1983, pp. 95-98. 3 Eiden, E., Eskay, R.L., Scott, J., Pollard, H. and Hotchkiss, A.J., Primary cultures of bovine chromaftin cells synthesize and secrete vasoactive intestinal polypeptide (VIP), Life Sci., 33 (1983) 687~i9Y 4 Fisher, S.K., Holz, R.W. and Agranoff, B.W., Muscarinic receptors in chromaffin cell cultures mediate enhanced phospholipid labeling but not catecholamine secretion, J. Neurochem., 37 (1981) 491 497. 5 Hokin, M.R., Benfey, B.G. and Hokin, L.E., Phospholipids and adrenaline secretion in guinea pig adrenal medulla, J. Biol. Chem.. 233 (1958) 81,~817. 6 lp, N.Y., Ho, C.K. and Zigmond, R.E., Secretin and vasoactive intestinal peptide acutely increase
319 tyrosine 3-monoxygenase activity in the rat superior cervical ganglion, Proc. Natl. Acad. Sci. USA, 79 (1982) 7566- 7569. 7 Kao, L.S. and Schneider, A.S., Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium, J. Biol. Chem., 260 (1985) 2019 2022. 8 Kawatani, M., Rutigliano, M. and deGroat, W.C., Depolarization and muscarinic excitation induced in a sympathetic ganglion by vasoactive intestinal polypeptide, Science, 229 (1985) 879 881. 9 Kelner, K.L., Levine, R.A., Morita, K. and Pollard, H.B., A comparison of trihydroxyindole and HPLC/electrochemical methods for catecholamine measurement in adrenal chromaffin cells, Neurochem. Int., 7 (1985) 373 378. l0 Linnoila, R.I., Diaugustine, R.P., Hervonen, A. and Miller, R.J., Distribution of (MetSI - and (Leu~)enkephalin, vasoactive intestinal polypeptide- and substance P-like immunoreactivities in human adrenal glands, Neuroscience, 5 (1980) 2247 2259. 11 Lundberg, J.M., Hedlund, B. and Bartfai, T , Vasoactive intestinal polypeptide enhances muscarinic ligand binding in cat submandibular salivary gland, Nature (London), 295 (1982) 147 149. 12 Misbahuddin, M., Isosaki, M., Houchi, H. and Oka, M., Muscarinic receptor-mediated increase in cytoplasmic free Ca 2+ in isolated bovine adrenal medullary cells: effect of TMB-8 and phorbol ester TPA, FEBS Lett., 190 (1985)25-28. 13 Oka, M., Isosaki, M. and Yanagihara, N., Isolated bovine adrenal medullary cells; studies on regulation of catecholamine synthesis and release. In E. Usdin et al. (Eds.), Catecholamine: Basic and Clinical Frontiers, Vol. 1, Pergamon, Oxford, 1979, pp. 7~72. 14 Said, S.I., Vasoactive intestinal polypeptide (VIP): current status, Peptides, 5 (1984) 143-150. 15 Schneider, A.S., Cline, H.T. and Lemaire, S., Rapid rise in cyclic GMP accompanies catecholamine secretion in suspensions of isolated adrenal chromaffin cells, Life Sci., 24 (1979) 1389 1394. 16 Tischler, A.S~, Perlman, R.L., Costopoulos, D. and Horwitz, J., Vasoactive intestinal peptide increases tyrosine hydroxylase activity in normal and neoplastic rat chromaffin cell cultures, Neurosci. Lett., 61 (1985) 141 146. 17 Trifaro, J.M., The effect of Ca ++ omission on the secretion of catecholamines and the incorporation of orthophosphate-32P into nucleotides and phospholipids of bovine adrenal medulla during acetylcholine stimulation, Mol. Pharmacol., 5 (1969) 420~431. 18 Yanagihara, N., Isosaki, M., Ohuchi, T. and Oka, M., Muscarinic receptor-mediated increase in cyclic GMP level in isolated bovine adrenal medullary cells, FEBS Lett., 105 (1979) 296-298.
315
Elsevier Scientific Publishers Ireland Ltd.
NSL 04325
Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells Mir Misbahuddin, Hitoshi Houchi, Atsushi Nakanishi, Kyoji Morita and Motoo O k a Department ofl Pharmacology, Tokushima University School of Medicine Tokushima (Japan) (Received 16 July 1986; Revised version received 2 September 1986; Accepted 3 September 1986)
Key wordsv Vasoactive intestinal polypeptide (VIP); Catecholamine secretion; Muscarinic receptor: Adrenal medullary cell; Guinea pig The effects of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion by isolated guinea pig adrenal medullary cells were studied. VIP (1 pM) alone induced only a slight secretion of CA, but it stimulated ACh-induced CA secretion. At concentrations of 0.01 l ,uM, it stimulated muscarine-induced CA secretion, but not nicotine-induced CA secretion. It did not affect high K + or Ca 2+ ionophore-induced CA secretion. The stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2 200/iM and was detectable after 2 min incubation.
The adrenal medulla is innervated by preganglionic sympathetic fibers from splanchnic nerves. Stimulation of these nerves causes release of acetylcholine (ACh) from the nerve terminals, which initiates the secretion of catecholamine (CA) from adrenal medullary cells. Recently, immunohistochemical studies demonstrated the presence of vasoactive intestinal polypeptide (VIP) not only in the splanchnic nerve terminals, but also in the adrenal medullary cells [2, 3, 10, 14]. These findings suggest that VIP is secreted with ACh from splanchnic nerve terminals or with CA from adrenal medullary cells, and modulates the secretion of CA mediated by ACh receptors. Therefore, we examined the effect of VIP on CA secretion from isolated guinea pig adrenal medullary cells. Results showed that VIP alone induced only a very slight CA secretion, but it significantly stimulated the secretion of CA induced by stimulation of muscarinic ACh receptors, but not that induced by stimulation of nicotinic ACh receptors. Guinea pigs (Hartley strain) of both sexes weighing 400 500 g were killed by a Correspondence. M. Oka, Department of Pharmacology, Tokushima University School of Medicine, 3 Kuramoto, T o k u s h i m a 770, Japan. 0304-3940,'86,,'$ 03.50 O 1986 Elsevier Scientific Publishers Ireland Ltd.
316
blow on the head and decapitation. In each experiment, 8-12 guinea pigs were killed and their adrenal glands were promptly removed. The medulla was separated from the adhering fat and the cortex with a pair of fine scissors, cut into several pieces and digested with collagenase as described elsewhere [13]. The isolated cells were suspended in Krebs-Ringer phosphate (KRP) buffer solution (in mM: NaC1 154, KCI 5.6, CaCI2 2.2, MgSO4 1.1, NaH2PO4 0.85, Na2HPO4 2.15, glucose 10 and 0.5% bovine serum albumin, pH 7.4) at a density of 5 to 7 x 105 cells/ml. The viability of the cells, determined by the Trypan blue exclusion test, was more than 95%. Bovine adrenal medullary cells, isolated by essentially the same method, were used in some experiments. The amount of CA released from the isolated adrenal medullary cells was measured by a modification of the trihydroxyindole method [9]. VIP was obtained from the Peptide Institute, Osaka, Japan. A peptidase inhibitor was not used. Table I shows the effects of VIP on basal CA secretion and CA secretion induced by the physiological secretagogue ACh, the nicotinic agonist nicotine and the muscarinic agonist muscarine. VIP alone at a concentration of I/zM induced only a slight secretion of CA, but it significantly stimulated ACh-induced CA secretion. It also stimulated muscarine-induced CA secretion dose-dependently, but did not stimulate nicotine-induced CA secretion. Its stimulatory effect on muscarine-induced CA secre-
TABLE I EFFECTS OF VIP ON BASAL AND INDUCED MEDULLARY CELLS
CA SECRETION FROM ISOLATED ADRENAL
The cells were preincubated (5 min, at 37°C) with or without VIP and then stimulated with ACh, muscarine, nicotine or high K + for 10 min or with A23187 for 30 min. After incubation, the cells were precipitated by centrifugation at 500 g for 5 min. CA in the cells and medium was extracted with 10% acetic acid and measured fluorometrically [9]. C A secretion was shown as a percentage of the total cellular C A content. The maximum secretion induced by each stimulant was expressed as 100%. Values are means __+S.E.M. of 3 ~ experiments. The effect of VIP was significant ( * P < 0 . 0 1 ) . Stimulant (/tM)
VIP (/zM)
CA secretion (% of total)
Stimulation (%)
-
1.0
--
ACh (200) ACh (200) Muscarine Muscarine Muscarine Muscarine
1.0 -0.01 0.1 1.0
1.3+0.2 2.5+0.4 7.8+0.3 11.3+0.4 6.3 + 0.4 7.7 + 0.5 8.5 + 0.5 9.2 _+0.6 4.6 _+0.3 5.7 + 0.6 6.0+0.5 5.9+0.5 9.6-t-0.8 10.1 + 0 . 8
(200) (200) (200) (200) N i c o t i n e (100) Nicotine (100) High K + (56 m M ) High K + (56 m M ) A23187 (10) A23187 (10)
1.0 1.0 1.0
100 145" 100 122 135* 146* 100 124
100 98
100 105
317
tion was detectable at 0.01 /tM and was significant at 0.1 and 1 /tM, the maximum concentration tested. We also examined the effect of VIP on CA secretion from isolated bovine adrenal medullary cells, from which secretion of CA is mainly induced by nicotinic stimulation. Results showed that VIP did not affect nicotine-induced CA secretion from isolated bovine adrenal medullary cells (data not shown). VIP also did not affect CA secretion caused by high K + and the Ca 2+ ionophore A23187 (Table 1). These results indicated that VIP has a selective stimulatory effect on muscarinic receptor-mediated CA secretion from isolated guinea pig adrenal medullary cells. As shown in Table II, the stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2-200/zM, with the highest concentration inducing the maximum response by itself. Fig. 1 shows the time course of stimulation by VIP of muscarine-induced CA secretion from isolated guinea pig adrenal medullary cells. Stimulation by VIP was detectable after 2 rain and persisted for at least 10 rain. It is interesting that VIP has a selective stimulatory effect on the muscarinic response. These results are consistent with recent electrophysiological findings that VIP potentiates muscarinic receptor-induced depolarization, but not nicotinic receptorinduced depolarization in superior cervical ganglionic cells [8]. The mechanism of the selective stimulatory effect of VIP on muscarinic responses is unknown. Secretion of CA by muscarinic stimulation from isolated guinea pig adrenal medullary cells was dependent on the presence of Ca 2+ in the medium, like the secretion of CA by nicotinic stimulation, suggesting that an increase in Ca 2+ uptake by the cells may be involved in the stimulation of CA secretion caused by muscarine. On the other hand, muscarinic stimulation is known to increase the cytoplasmic level of free Ca 2 +, which may be released from store sites [7, 12], increase phosphatidyl inositol turnover (the so-called PI response) [1, 4, 5, 17] and increase the cyclic G M P level in the cells [15, 18]. VIP may stimulate these responses and thereby muscarinic receptor-mediated CA secretion from the cells. In studies on a membrane preparation from the cat salivT A B L E I1 E F F E C T S O F VIP O N C A S E C R E T I O N I N D U C E D BY D I F F E R E N T
FROM
CONCENTRATIONS
ISOLATED ADRENAL
MEDULLARY
CELLS
OF MUSCARINE
The cells were i n c u b a t e d as described in T a b l e 1. C A secretion was s h o w n as a p e r c e n t a g e of the total C A in the cells. C A secretion induced by m u s c a r i n e w i t h o u t VIP was expressed as 100%. Values are the m e a n s o f 3 experiments. Muscarine (llM)
VIP (/~M)
C A secretion (% o f total)
Stimulation (%)
2 2 20 20 200 200
-1 -1
0.9 1.5 3.3 4.8 5.0 7.8
t00 166 100 149 100 156
I
318
10tO x-
O A O
.E ~E
5-
o
r, c,
£b 05
0 Time
10
(rain)
Fig. 1. Time course of stimulatory effect of VIP on muscarine-induced CA secretion from isolated adrenal medullary cells. Cells were incubated for various periods with musearine (200 #M) (O) or with muscarine (200/~M) plus VIP (1 ~tM) (0). CA secretion was shown as a percentage of the total cellular CA content. Points are means of two separate experiments.
ary gland, VIP was found to increase the binding of the muscarinic agonist [3H]Nmethyl-4-piperidinyl benzilate to membrane receptors [11]. Recently, Ip et al. [6] and Tischler et al. [16] found that VIP stimulated the synthesis of CA from tyrosine, by activation of tyrosine hydroxylase in rat sympathetic ganglion cells and rat PC 12 cells. In rat PC 12 cells, VIP increased the level of cyclic AMP [16], which activates tyrosine hydroxylase. In this experiment, the effect of VIP on the cyclic AMP level in isolated guinea pig adrenal medullary cells was not examined. However, since VIP stimulated muscarine-induced CA secretion from the cells whereas dibutyryl cyclic AMP did not (data not shown), it does not seem to stimulate muscarine-induced CA secretion by a mechanism involving cyclic AMP. Further studies are required on the mechanism of the effect of VIP in stimulating the muscarinic response.
We thank Mrs. Keiko Tachibana for typing this manuscript. This work was supported by grants from the Japanese Ministry of Education, Science and Culture. 1 Azila, N. and Hawthorne, J.N., Subcellular localization of phospholipid changes in response to muscarinic stimulation of perfused bovine adrenal medulla, Biochem. J., 204 (1982) 291--299. 2 Carmichael, S.W., The Adrenal Medulla, Vol. 3, Eden Press, Quebec, 1983, pp. 95-98. 3 Eiden, E., Eskay, R.L., Scott, J., Pollard, H. and Hotchkiss, A.J., Primary cultures of bovine chromaftin cells synthesize and secrete vasoactive intestinal polypeptide (VIP), Life Sci., 33 (1983) 687~i9Y 4 Fisher, S.K., Holz, R.W. and Agranoff, B.W., Muscarinic receptors in chromaffin cell cultures mediate enhanced phospholipid labeling but not catecholamine secretion, J. Neurochem., 37 (1981) 491 497. 5 Hokin, M.R., Benfey, B.G. and Hokin, L.E., Phospholipids and adrenaline secretion in guinea pig adrenal medulla, J. Biol. Chem.. 233 (1958) 81,~817. 6 lp, N.Y., Ho, C.K. and Zigmond, R.E., Secretin and vasoactive intestinal peptide acutely increase
319 tyrosine 3-monoxygenase activity in the rat superior cervical ganglion, Proc. Natl. Acad. Sci. USA, 79 (1982) 7566- 7569. 7 Kao, L.S. and Schneider, A.S., Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium, J. Biol. Chem., 260 (1985) 2019 2022. 8 Kawatani, M., Rutigliano, M. and deGroat, W.C., Depolarization and muscarinic excitation induced in a sympathetic ganglion by vasoactive intestinal polypeptide, Science, 229 (1985) 879 881. 9 Kelner, K.L., Levine, R.A., Morita, K. and Pollard, H.B., A comparison of trihydroxyindole and HPLC/electrochemical methods for catecholamine measurement in adrenal chromaffin cells, Neurochem. Int., 7 (1985) 373 378. l0 Linnoila, R.I., Diaugustine, R.P., Hervonen, A. and Miller, R.J., Distribution of (MetSI - and (Leu~)enkephalin, vasoactive intestinal polypeptide- and substance P-like immunoreactivities in human adrenal glands, Neuroscience, 5 (1980) 2247 2259. 11 Lundberg, J.M., Hedlund, B. and Bartfai, T , Vasoactive intestinal polypeptide enhances muscarinic ligand binding in cat submandibular salivary gland, Nature (London), 295 (1982) 147 149. 12 Misbahuddin, M., Isosaki, M., Houchi, H. and Oka, M., Muscarinic receptor-mediated increase in cytoplasmic free Ca 2+ in isolated bovine adrenal medullary cells: effect of TMB-8 and phorbol ester TPA, FEBS Lett., 190 (1985)25-28. 13 Oka, M., Isosaki, M. and Yanagihara, N., Isolated bovine adrenal medullary cells; studies on regulation of catecholamine synthesis and release. In E. Usdin et al. (Eds.), Catecholamine: Basic and Clinical Frontiers, Vol. 1, Pergamon, Oxford, 1979, pp. 7~72. 14 Said, S.I., Vasoactive intestinal polypeptide (VIP): current status, Peptides, 5 (1984) 143-150. 15 Schneider, A.S., Cline, H.T. and Lemaire, S., Rapid rise in cyclic GMP accompanies catecholamine secretion in suspensions of isolated adrenal chromaffin cells, Life Sci., 24 (1979) 1389 1394. 16 Tischler, A.S~, Perlman, R.L., Costopoulos, D. and Horwitz, J., Vasoactive intestinal peptide increases tyrosine hydroxylase activity in normal and neoplastic rat chromaffin cell cultures, Neurosci. Lett., 61 (1985) 141 146. 17 Trifaro, J.M., The effect of Ca ++ omission on the secretion of catecholamines and the incorporation of orthophosphate-32P into nucleotides and phospholipids of bovine adrenal medulla during acetylcholine stimulation, Mol. Pharmacol., 5 (1969) 420~431. 18 Yanagihara, N., Isosaki, M., Ohuchi, T. and Oka, M., Muscarinic receptor-mediated increase in cyclic GMP level in isolated bovine adrenal medullary cells, FEBS Lett., 105 (1979) 296-298.