Vasoactive Intestinal Polypeptide and Muscarine Mobilize Intracellular Ca2+ through Breakdown of Phosphoinositides to Induce Catecholamine Secretion ROLE OF IPa IN EXOCYTOSIS*

We have recently shown that vasoactive intestinal polypeptide (VIP) is as potent as acetylcholine in in- ducing the secretion of catecholamines from the rat adrenal medulla. In the present study we have inves- tigated the molecular mechanism involved in the exocytotic secretion of catecholamines by VIP and the effects of VIP on Ca4' uptake and phosphoinositide breakdown and compared them with those of the classical cholinergic agonists. We now show that omission of Ca" from the perfusion medium had almost no effect on VIP-induced secretion; however, addition of 1 mM EGTA to calcium-free medium abolished the secretion. Stimulation with VIP did not result in a net increase in Ca4' uptake and it was not modified by a protein kinase C activator, phorbol ester. All these effects of VIP were comparable to those of muscarine. VIP (0.3 to 10 WM) and muscarine (30 to 100 WM) produced time-and concentration-dependent increase (up to 700%) in the production of ["Hlinositol phosphates. The production of ["Hlinositol phosphates by VIP and muscarine occurred in calcium-free and EGTA medium. The effect of VIP on ["HIIP, ["H]IPz, and ["HIIP, Model LS7000). Total counts after correction for dilution and quench- ing were converted to picograms and expressed as picograms/milli-grams of the wet weight of the adrenal medulla. Inositol Phosphates-Our unpublished results have firmly established the integrity of the isolated adrenal medulla kept in Krebs solution for several hours. We measured lactate dehydrogen- ase and catecholamine contents of the adrenal medulla maintained in uitro for 4 h and found that there was virtually no change in the contents compared with those of the freshly isolated medullae (1.43 f 0.05 and 1.41 t 0.2 pg/g for catecholamines and 175 5 19 and 167 f 25 units/mg for lactate dehydrogenase). Therefore, for the sake of convenience and efficiency most of the experiments involving the measurements of inositol phosphate were carried on isolated adrenal medullae. The medulla of the right and left adrenal glands was separated from the cortical tissues at 4 "C and incubated in 0.5 ml of Krebs solution containing 10 pl of [3H]myo-inositol (specific activity 17.1 Ci/mmol) for 60 min at 37 "C with vigorous shaking in a Dubonoff shaker. The tissue was washed with Krebs solution for 15 min and then with 10 mM lithium/Krebs for another 15 min. The gland was then incubated in 0.25 ml of lithium/Krebs or lithium/Krebs con- taining various agents for different times. The reaction was termi-nated by adding 0.5 ml of 10% trichloroacetic acid. The tissue was homogenized and centrifuged (9500 rpm) for 15 min. The pellet was used for protein estimation (15). The supernatant was neutralized with NaOH and placed on a resin (AG 1-X8, 200-400 mesh formate form). Different [3H]phosphoinositols were eluted from the column as described before (16). The radioactivity in each 5-ml eluate was determined in a liquid scintillation counter and was expressed as

We have recently shown that vasoactive intestinal polypeptide (VIP) is as potent as acetylcholine in inducing the secretion of catecholamines from the rat adrenal medulla. In the present study we have investigated the molecular mechanism involved in the exocytotic secretion of catecholamines by VIP and the effects of VIP on Ca4' uptake and phosphoinositide breakdown and compared them with those of the classical cholinergic agonists. We now show that omission of Ca" from the perfusion medium had almost no effect on VIP-induced secretion; however, addition of 1 mM EGTA to calcium-free medium abolished the secretion. Stimulation with VIP did not result in a net increase in Ca4' uptake and it was not modified by a protein kinase C activator, phorbol ester. All these effects of VIP were comparable to those of muscarine. VIP (0.3 to 10 WM) and muscarine (30 to 100 WM) produced timeand concentration-dependent increase (up to 700%) in the production of ["Hlinositol phosphates. The production of ["Hlinositol phosphates by VIP and muscarine occurred in calcium-free and EGTA medium. The effect of VIP on ["HIIP, ["H]IPz, and ["HIIP, production was reduced by (1 to 30 PM) VIP antagonist (an analogue of growth hormone-releasing factor, Ac-Tyr'hGRF) and 1 to 20 PM naloxone. Although nicotine produced a brisk secretory response, there was no change in ["Hlinositol phosphates. We conclude that inositol 1,4,5-trisphosphate generated upon activation of VIP and muscarine receptors is linked to exocytotic secretion of adrenal medullary hormones through release of internal Ca2+ ions.
Chromaffin cells of the rat adrenal medulla can be stimu-*This work was supported by Grants HL-22170 and HL-18601 from the National Institutes of Health. The work represents part of the research carried out by R. K. M. in fulfilling the requirements for the degree of Doctor of Philosophy in the Graduate School of the State University of New York, Brooklyn, NY. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lated through activation of presynaptic splanchnic nerve terminals that release the classical neurotransmitter, acetylcholine (1). Acetylcholine activates nicotinic as well as muscarinic receptors to induce secretion of catecholamines (2). Activation of nicotinic receptors results in mobilization of extracellular Ca'+, whereas activation of muscarinic receptors mobilizes intracellular Ca2+ which is subsequently used for secretion of catecholamines (3,4). Recent evidence suggests that influx of Ca2+ initiated by stimulation of nicotinic receptors is regulated by protein kinase C (4). Events that regulate mobilization of intracellular Ca2+ after stimulation of muscarinic receptors are not yet resolved. Several investigators have shown that stimulation of muscarinic receptors of bovine adrenal chromaffin cells and PC12 cells releases Ca2+ from intracellular sites to raise free cytosolic concentration of Ca'+ (5, 6). The mobilization of internal Ca'+ is mediated by a second messenger, IP31 (6-8). However, the increase in free cytosolic Ca'+ is of insufficient magnitude to induce exocytosis (9, 10). Therefore, the physiological role of IP, in exocytosis still remains unresolved, at least in the secretion of adrenal catecholamines. In the present study we have investigated the effects of muscarine, nicotine, and VIP on generation of inositol phosphates, mobilization of Ca'+, and secretion of catecholamines. The reason for selecting VIP was as follows.
Our recent studies have indicated that presynaptic nerve terminals liberate excitatory substance(s) in addition to acetylcholine to trigger the secretion of catecholamines from the rat adrenal gland (11). Although our search has not succeeded in identifying the exact nature of the noncholinergic substance, VIP appears to be the most attractive candidate among various peptides that are present in the splanchnic nerve endings. For one reason, VIP is as potent as acetylcholine in evoking the secretion of catecholamines (12). Since VIPevoked secretion is dependent on the presence of Ca2+, it was of great interest to determine the source of Ca2+ (intraor extracellular) and possibly the role of second messengers (diacylglycerol or IPS) in mobilization of Ca'+ in exocytosis. We report here that VIP, like muscarine, has no effect on the influx of extracellular Ca2+, but enhances intracellular mobilization of Ca'+ through generation of IP, to induce catecholamine secretion. We also show that phosphoinositide hydrolysis by VIP and muscarine occurs in calcium-free plus 1 mM EGTA medium.

Retrograde Perfusion of the Adrenal Gland and Perfusion Media-
The left adrenal gland of the male rat (300-400 g) was perfused retrogradely, at 0.35 ml/min with Krebs bicarbonate solution, as described previously (13). The composition of the solution was as follows (mM): NaC1, 119; KC], 4.7; CaC12, 2.5; MgSO,, 1.2; glucose, 11; and NaHC03, 25. In addition, the solution contained 0.027 mM Na2EDTA to prevent oxidation of catecholamines. The solutions were constantly bubbled with 95% O2 plus 5% CO, to maintain the pH at 7.4. Lithium/Krebs was made by adding desired amounts of LiCl to Krebs solution and removing equivalent molar amounts of NaCI. Adrenal glands were stimulated before and after perfusion with

Phosphoinositide Hydrolysis by VIP and Muscarine
LiCl/Krebs solution by directly injecting 0.3 pg of nicotine, 30 pg of muscarine, and 10 pg of VIP into the perfusion stream. Measurement of Catechohmines-Catecholamine content of the perfusate was analyzed directly by the fluorometric method (14) without the intermediate purification on alumina for the reasons given earlier (13). The content of catecholamines in the perfusate was expressed in terms of epinephrine base. Amounts of catecholamines secreted during the nonstimulation period have been subtracted from those secreted in the stimulation period to obtain net secretion. The amount of catecholamines secreted in the 5-min nonstimulation period was 6 t 3 ng (n = 22).
Measurement of Ca4s Uptake-The uptake of Ca4' was measured in the rat adrenal medulla as described before (4). The adrenal gland was perfused with Krebs solution for 30 min. The medium was then changed to Krebs solution containing 0.5 mg/ml Ca45 (as 45CaC12, specific activity 23.69 mCi/mg) for 10 min. The gland was perfused for 30 min with ice-cold Krebs solution. An identical protocol was carried out in other adrenal glands, except that the 10-min perfusion with Ca" Krebs solution contained various secretagogues. In another series of experiments the above protocol was carried out, except that before exposure to Ca45 medium the adrenal gland was perfused with Krebs solution containing 30 nM phorbol12,13-dibutyrate for 15 min. Phorbol ester was also present during 10-min perfusion with Ca45 medium alone, or that containing a stimulatory agent. After the 30min wash period, the adrenal gland was removed and the adrenal medulla was separated, weighed, and homogenized in 1 ml of 0.05 M perchloric acid; the homogenate was centrifuged and the supernatant (0.5 ml) was counted in a liquid scintillation counter (Beckman, Model LS7000). Total counts after correction for dilution and quenching were converted to picograms and expressed as picograms/milligrams of the wet weight of the adrenal medulla.
Analysis of Inositol Phosphates-Our unpublished results have firmly established the integrity of the isolated adrenal medulla kept in Krebs solution for several hours. We measured lactate dehydrogenase and catecholamine contents of the adrenal medulla maintained in uitro for 4 h and found that there was virtually no change in the contents compared with those of the freshly isolated medullae (1.43 f 0.05 and 1.41 t 0.2 pg/g for catecholamines and 175 5 19 and 167 f 25 units/mg for lactate dehydrogenase). Therefore, for the sake of convenience and efficiency most of the experiments involving the measurements of inositol phosphate were carried on isolated adrenal medullae.
The medulla of the right and left adrenal glands was separated from the cortical tissues at 4 "C and incubated in 0.5 ml of Krebs solution containing 10 pl of [3H]myo-inositol (specific activity 17.1 Ci/mmol) for 60 min at 37 "C with vigorous shaking in a Dubonoff shaker. The tissue was washed with Krebs solution for 15 min and then with 10 mM lithium/Krebs for another 15 min. The gland was then incubated in 0.25 ml of lithium/Krebs or lithium/Krebs containing various agents for different times. The reaction was terminated by adding 0.5 ml of 10% trichloroacetic acid. The tissue was homogenized and centrifuged (9500 rpm) for 15 min. The pellet was used for protein estimation (15). The supernatant was neutralized with NaOH and placed on a resin (AG 1-X8, 200-400 mesh formate form). Different [3H]phosphoinositols were eluted from the column as described before (16). The radioactivity in each 5-ml eluate was determined in a liquid scintillation counter and was expressed as counts/min/mg of protein. All data are presented as mean f standard errors, and statistical comparisons were made by Student's t test.

RESULTS AND DISCUSSION
Fig. la shows that omission of CaClz from the perfusion medium for 30 min did not significantly affect the secretion evoked by VIP. The amounts of catecholamines secreted in calcium-free medium were 88% of those in the normal medium. If 1 mM EGTA was added to calcium-free Krebs solution in order to reduce the intracellular stores of Ca2+ in chromaffin cells, VIP was ineffective (only 10% of control) in evoking the secretion. Readdition of CaC12 (2.5 mM) partially restored the stimulatory action of VIP. Table I shows the relationship between Ca45 accumulation and catecholamine secretion evoked by VIP a n d cholinergic agonists before and after treatment with phorbol 12,13-dibutyrate. As reported earlier for muscarine

TABLE I
Inability of VIP to mobilize Ca45 in the absence and presence of phorbol ester The experimental protocol was identical to that described earlier (4) for measuring catecholamine secretion and Ca45 accumulation after subjecting the adrenal medulla to various experimental procedures. Each agent was introduced along with Ca4' for 10 min. The perfusate collected in this period was assayed for catecholamines, and the medulla was removed 30 min after washout of Ca4' to estimate Ca4' accumulation. The same procedure was repeated in other experiments, except that the gland was pretreated with phorbol 12,13dibutyrate for 15 min prior to exposure to various agents. Each value represents a mean of four to six observations.

Phosphoinositide Hydrolysis by VIP and Muscarine 2125
The above results ( Fig. 1 and Table I) indicated that VIP mobilizes mostly intracellularly bound Ca2+ for inducing catecholamine secretion, and Ca2+ mobilization may involve hydrolysis of membrane phospholipids. Lithium chloride is known to increase the accumulation of inositol phosphates by inhibiting inositol 1-phosphatase (17). Therefore, effects of LiCl were tested on secretion induced by VIP, muscarine, and nicotine. As shown in Fig. lb, 10 mM LiCl produced a significant facilitation (50%) of catecholamine secretion evoked by VIP and muscarine. The effect was further enhanced (100 to 200%) when LiCl concentration was raised to 20 and 30 mM. The same figure shows that only very high concentrations of LiCl(20 and 30 mM) had a facilitatory effect (about 50%) on secretion evoked by nicotine. None of the concentrations of lithium affected the basal secretion of catecholamines (not shown).
3 p~ muscarine caused a significant rise in the production of [3H]inositol phosphates, [3H]IP being the most prominent product. The production of [3H]IP (Fig. 2a) increased over 6fold when the concentration of muscarine was raised to 100 p~. The other products, ['H]IP, and [,H]IP3, increased by about 2-fold with 30 p~ muscarine. Fig. 26 shows that as low as 0.3 ptM VIP produced a significant increase in the generation of [3H]IP, [3H]IP2, and [3H]IP3, and the amounts of all the products increased with an increase in VIP concentration. Although not shown, the incubation medium contained large quantities of catecholamines after exposing the adrenal medulla to various concentrations of muscarine and VIP. Fig. 3 shows the time course of production of [3H]inositol phosphates when the adrenal medulla is incubated with a fixed concentration of muscarine and VIP for varying periods of time. Within the first 2 min of incubation, the most prominent product of phosphoinositide hydrolysis was [,HI IP,. The other products were only marginally increased in 2 min. There was a fall in [3H]IP3 production between 2 to 5 min and a sharp rise in [3H]IP production, which increased up to 700% in the next 25 min. The production of [3H]IP3 remained at 60 to 90% of the control throughout. The insets of Fig. 3, A and B show that muscarine-and VIP-evoked secretion of catecholamines, which reached a peak in 5 to 10 min, remained at that level during the next 25 min without a decline in the secretion. Fig. 3A also shows that nicotine, which is a powerful secretogogue (Table I), did not increase production of ['HIIP. In all the above experiments, the secretion of catecholamines was measured from the perfused adrenal glands, and the [3H]inositol phosphates were estimated in isolated adrenal medullae. Therefore, one may argue about the role of [3H]IP,, as a second messenger, in the secretion of catecholamines from the perfused adrenal gland. To resolve this crucial point, in several experiments phosphoinositide hydrolysis was estimated in the perfused adrenal preparations before and after exposing to 100 p~ muscarine. The adrenal gland was loaded with [3H]inositol (30 pl in 3 ml of Krebs) by recirculating the solution for 1 h followed by washout with Krebs solution for 45 min and then 15 min with LiCl/Krebs solution. The control glands were removed after 2 min and the medulla was separated and then processed for estimation of [3H]inosit~l phosphates. The experimental glands were perfused with 100 p M muscarine for 2 min and then removed for the analysis. The respective values of ['HJIP,and [3H]IP were 78 f 6 and 804 f 38 cpm/mg protein, n = 5 , for control, and 146 f 8 and 1138 f 56 cpm/mg protein, n = 5 , for muscarine. During 2 min perfusion with muscarine, the amounts of catecholamines secreted were 112 f 8 ng as compared to the control (6 f 2 ng, n = 5 ) . These experiments clearly establish a true link between ['HIIP, and catecholamine secretion under identical experimental conditions. We examined the need of Ca2+ in the production of inositol phosphates by muscarine and VIP. If muscarine and VIP were tested in the adrenal medulla which was bathed in calciumfree and 1 mM EGTA/Krebs solution, the amounts of [3H]IP produced by both agonists were almost identical to those produced in calcium-containing medium (560 f 80% uersus 495 2 70% for 100 p M muscarine and 195 f 50% uersus 180 f 30% for 3 pM VIP, n = 4).
Recently we have reported that naloxone (3 to 30 p~) interferes with the secretion of catecholamines evoked by VIP (12). Therefore, it was decided to test naloxone on VIPinduced breakdown of phosphoinositides. Experiments were also extended to Ac-Tyr'hGRF, a competitive antagonist of VIP (18). As shown in Fig. 4a, increasing concentrations of naloxone produced a decline in the production of [3H]in~~itol phosphates. 20 p~ naloxone almost abolished the effect of VIP. Similarly Ac-Tyr'hGRF produced a concentration-dependent decrease but the effect was less prominent than that of naloxone (Fig. 4b).
The present results clearly show that, among three secre- tagogues, muscarine and VIP but not nicotine increase the hydrolysis of phosphoinositides. The hydrolysis is associated with an increase in production of inositol phosphates and the secretion of catecholamines. Our previous experiments have already established that VIP stimulates catecholamine secretion by directly acting on chromaffin cells and not via release of acetylcholine (12). Here we show that VIP-induced hydrolysis of phosphoinositides was reduced by naloxone and a specific antagonist of VIP. These observations lend further support to the proposal that VIP activates its own receptors to trigger phosphoinositides to mobilize Ca2+ and thereby induce exocytotic secretion of catecholamines. Previous reports on the effects of VIP on inositol phospholipid metabolism in the superior cervical ganglia of rat are contradictory (19,20).
It should be mentioned that VIP is known to increase cAMP content in different tissues (22, 23). Therefore, a question arises whether a rise in cAMP is somehow related to the current findings. We do not know the answer. However, forskolin does not induce catecholamine secretion from the rat adrenal gland nor has it any effect on phospholipid hydrolysis (24).' Several workers have shown that activation of muscarinic receptors increases the hydrolysis of phosphoinositides to mobilize intracellular Ca2+ from a nonmitochondrial source (6)(7)(8)21). However, the consequence of Ca2+ mobilization, and therefore the physiological role of IP3, had remained unexplained. This gap is now fulfilled by the present investigation. We have shown not only that muscarinic receptor activation enhances the production of [3H]inositol phosphates, but that such a n action is followed by secretion of catecholamines. Muscarinic stimulation for as long as 30 min resulted in sustained secretion of catecholamines and was associated with an increase in the production of [3H]inositol phosphates. It was particularly interesting to observe a close relationship between production of ['HIIP, and secretion of catecholamines within the first 5 min. As the incubation was continued for 30 min, ['H]1P3 production as well as catecholamine secretion leveled off. These observations help in understanding the lack of desensitization after prolonged exposure of the adrenal gland to muscarine. If there is an uninterrupted production of [3H]IP3 during continuous exposure to muscarine and if there is a large storage of internal Ca2+, it is not surprising that the secretory response would remain elevated as long as muscarine stimulates the receptors of the adrenal medulla. The actions of VIP, both on production of inositol phosphates and catecholamine secretion, very closely resembled those of muscarine in almost every aspect. Our studies also provide clear evidence that hydrolysis of phosphoinositides occurs in calcium-free and 1 mM EGTA medium where the secretory response is almost abolished. It is likely that Ca2+ is not essential for the breakdown of membrane phospholipids by muscarine and VIP.
Based on the present and our earlier observation, it is possible to describe the physiological role of various receptors of chromaffin cells in the secretion of catecholamines. Activation of muscarinic and nicotinic receptors by acetylcholine released from nerve terminals triggers two pathways for mobilization of Ca2+ to raise its free concentration in cytosol.
During prolonged exposure to acetylcholine, as in stress, nicotinic receptors desensitize but muscarinic receptors do not (Fig. 3); which allows an uninterrupted secretion of catecholamines. In addition, if VIP is released as a noncholinergic transmitter (11, 12), its contribution in the total secretory response would be highly valuable during stress, especially when nicotinic receptors tend to lose their functional role.