Vasoactive intestinal polypeptide increases inositol phospholipid breakdown in the rat superior cervical ganglion

Brain Research, 376 (1986) 363-367

363

Elsevier BRE 21594

Vasoactive intestinal polypeptide increases inositol phospholipid breakdown in the rat superior cervical ganglion SYLVIE AUDIGIER, CLAUDE BARBERIS and SERGE JARD Centre C. N. R. S. -I. N. S. E. R. M. de Pharmacologie-Endocrinologie, Montpellier (France)

(Accepted February 18th, 1986) Key words: vasoactive intestinal polypeptide (VIP) receptor - - inositol phosphate - - sympathetic ganglion

The effects of VIP and of peptides of the VIP family: secretin, glucagon, the porcine histidine isoleucine containing peptide (PHI) and the rat hypothalamic growth hormone-releasing hormone (rhGRF) on the cyclicAMP and inositol phosphate contents of isolated rat superior cervical ganglia were investigated. We demonstrate that VIP is able to provoke a large inositol lipid breakdown by acting directly on ganglionic cells. This observation suggests the presence in rat superior cervical ganglia of a new type of receptors for VIP or for an unidentified peptide structurally related to VIP.

For a long time, acetylcholine has been thought to be the sole neurotransmitter in sympathetic ganglia. Several lines of evidence now indicate that other substances, mainly a variety of neuropeptides, may also act as transmitters in these ganglia 9-11'15'17'19. Some of these transmitters, like arginine vasopressin and muscarinic agonists were shown to stimulate inositol lipid breakdown in isolated rat superior cervical ganglion 3A3, suggesting an action on calcium-dependent regulatory pathways of neuronal function. Noradrenaline, on the other hand, strongly stimulates cyclic AMP formation s . Therefore, the rat superior cervical ganglion provides a good system with which to study possible interactions between cyclic AMP- and calcium-dependent regulatory pathways of neuronal function. For instance, changes in phospholipid metabolism are known to mediate the activation of tyrosine 3-monooxygenase (Tyr(OH)ase) by muscarinic agonists 12. Secretin and vasoactive intestinal polypeptide (VIP) also acutely increase Tyr(OH)ase activity in the superior cervical ganglion 14. Since secretin and VIP receptors are coupled to adenylate cyclase in all tissues so far studied 7'23, the above observations suggest either that both calcium and cyclic AMP are involved in the regulation of Tyr(OH)ase in

the rat superior cervical ganglion, or that, in this particular tissue, secretin and VIP promote calcium mobilization through increased inositol lipid breakdown. That VIP plays a functional role in sympathetic ganglia is also suggested by the fact that VIP enhances the sensitivity of postsynaptic muscarinic receptors to acetylcholine in guinea pig inferior mesenteric ganglia 16. The effect of VIP and other agents tested on inositol lipid breakdown was assayed by measuring the accumulation of labelled inositol phosphates in [3H]inositol-prelabelled ganglia incubated in the presence of Li ÷, as previously described 3. Superior cervical ganglia were dissected from male Wistar rats (200-250 g). The afferent and efferent nerves were cut off and the connective tissue sheaths removed. They were incubated for 2 h at 37 °C in a shaking incubator in 6 ml of Krebs-Ringer bicarbonate medium (composition in mM: NaC1 125, KCI 3.5, KH2PO 4 1.25, Mg SO 4 1.2, CaC12 0.75, N a H C O 3 25, glucose 10) containing 200/aCi [2-3H(N)]myoino sitol 16.5 Ci/mmol (New England Nuclear, Boston, MA) gassed with an atmosphere composed of 95% 02 and 5% CO2. The ganglia were then rinsed with the same medium containing no inositol. Each gan-

Correspondence: S. Audigier, Centre C.N.R.S.-I.N.S.E.R.M. de Pharmacologie-Endocrinologie, Rue de la Cardonille - - B.P. 5055, 34033 Montpellier Cedex, France.

0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

364 glion was transferred to a tube containing 230 ,ul of prewarmed, oxygenated Krebs-Ringer bicarbonate. After 15 min, 10 mM LiCI was added so as to inhibit the hydrolysis of inositol phosphates formed 2. 20 min later, the appropriate agonist was added and incubation usually continued for 20 min. incubations were terminated by the addition of 12.5/A of 70% (w/v) HCIO 4. The ganglia were homogenized. The acid extract was neutralized with 1.5 M K O H and buffered with 75 mM HEPES. The inositol phosphates were separated by ion exchange chromatography on Dowex-1 × 10 columns as described in ref. 3. Lipids were extracted from the acidtreated ganglia with chloroform/methanol/12 M HC1 (100:200:1, by vol.). The solvent was evaporated and the incorporation of [3H]inositol into the lipids was determined. Relative intracellular cyclic AMP levels were determined by measuring the formation of tritiated cyclic AMP from the prelabelled adenine nucleotide pool. Ganglia were desheathed and incubated at 37 °C in 6 ml of Krebs-Ringer bicarbonate medium containing 24/~Ci [3H]adenine (24 Ci/mmol; Radiochemical Center, Amersham, U.K.) gassed with an atmosphere composed of 95% 02 and 5% CO 2. The ganglia were then washed and incubated individually in tubes containing 240 #1 of Krebs-Ringer bicarbonate in the presence of isobutylmethylxanthine, 1 mM. The agents tested were added 15 min later. After a further 20 rain incubation period the reaction was terminated by adding 250/tl of 10% TCA. ATP and cyclic AMP were added to the acid extracts at a

concentration of 0.5 mM. Labelled cyclic AMP was separated by sequential chromatography on Dowex and alumina columns as described in ref. 20. Radioactivity present in the cyclic AMP fraction was expressed as % of the sum of radioactivity recovered in the cyclic AMP fraction and radioactivity which was not retained by the Dowex column. The latter mainly corresponds to labelled ATP. The results presented here come from experiments with ganglia prelabelled for 2 h with [3H]myoinositol. In the majority of experiments, the incorporation of labelled inositol into the lipids of a single ganglion varied within fairly large limits: from 50,000 to 150,000 dpm with a mean value of about 100,000 dpm. Accordingly, the radioactivity of each phosphate ester also varied. Homogeneous data were obtained when the radioactivity found in inositol phosphates was expressed as a fraction of total radioactivity found in inositol lipids. This observation suggests that the variability in inositol lipid labelling more likely reflects the influence of factors like ganglion size or metabolic activity state rather than differences in the processes involved in inositol lipid breakdown and its regulation. In line with previously reported results 3, we found that carbachoi (10/~M) and arginine vasopressin (100 nM) produced a marked increase in the inositol phosphate content of rat superior cervical ganglia (Table I). Unexpected was the observation that VIP (8/~M) also enhanced inositol lipid breakdown. The magnitude of the response to VIP stimulation (a 5-fold increase over basal value) represented about a third of

TABLE I Effects of muscarinic vasopressin and angiotensin antagonists on VIP-induced inositol phosphate accumulation by rat superior cervical ganglia

The antagonists were added at the same time as the agonists. Atropine, d(CH2)5[Tyr(Et)2,Val4,o-ArgS]-vasopressin,and [Sarm,IleS]angiotensin II were used at concentrations of i raM, 1/zM, and 140 nM respectively. VIP, arginine vasopressin, and angiotensin were purchased from Bachem (Switzerland). Data in the table are means _+S.D. of the number of determinations indicated in brackets. Agent tested

None VlP (8 #M) Carbachol (10/~M) Arginine vasopressin (10 nM) Arginine vasopressin (100 nM) Angiotensin II (5 nM)

[3H] Inositol phosphates content (% of lipids) Without

With specific antagonist

4.3 + 0.9 (10) 20.7 + 3.7 (7) 20.7 + 3.7 (7) 20.7 + 3.7 (7) 46.2 + 5.7 (5) 9.0 + 1.9 (3) 42.9 + 1.8 (3) 7.7 _+ 1.0 (3)

15.1 + 3.7 (3) (atropine) 21.9 +__2.4 (3) (VP antagonist) 25.6 (AI1 antagonist) 3.9 + 0.8 (3) (atropine) 4.0 _+0.4 (3) (VP antagonist) 18.8 + 2.9 (3) (VP antagonist) 5.0 (AII antagonist)

365 that elicited by carbachol or arginine vasopressin. At variance with a previous report 4 we also found that angiotensin II was active in eliciting inositol phosphate accumulation by rat superior cervical ganglia (Table I). In all experimental conditions explored, no marked differences in the relative proportions of InsP, InsP2 and InsP 3 were observed. InsP, InsP2 and InsP 3 represented 62.8 + 3.4, 28.5 + 2.0 and 8.6 + 1.6% (28 determinations) of total inositol phosphates respectively. The effects of VIP, arginine vasopressin, angiotensin, and carbachol seem homologous. It is tempting to conclude from these results that all the agents tested induced calcium mobilization in their target cells through a receptor-mediated increase in Ins, 1,4,5P3 production. Further experiments would be needed to ascertain this conclusion. Indeed: (1) it was shown in several systems that the production of Ins 1,3,4P 3 (inactive as a calcium mobilizing agent) is also increased in stimulated cells; (2) the two inositol phosphates are not separated by the chromatographic procedure used in the present study. Since the experiments were conducted using intact isolated ganglia it was not possible, at that level, to decide if the effect of VIP on inositol phosphate formation resulted from a direct action of the VIP on inositol lipid breakdown in a VIP-sensitive cell population, or indirectly via a VIP-induced liberation of one or more neurotransmitters that mediate an inositol lipid breakdown in other cell populations. As shown in Table I, the responses to carbachol, vasopressin, and angiotensin could be inhibited by atropine, d(CH2)5[Tyr(Et)]VDAVP, and [Sarl,Ile8]-an giotensin II respectively. None of these antagonists blocked the response to VIP, indicating that this response was not a consequence of VIP-induced liberation of endogenous acetylcholine, vasopressin or angiotensin. In a second series of experiments we measured the response to VIP under experimental conditions known to block neurotransmitter release by nerve terminals (elimination of extracellular calcium, or presence of tetrodotoxin (TTX)). As shown in Fig. 1 depolarization induced by elevating extracellular potassium did increase inositol lipid breakdown. This effect, which very likely represents the combined actions of transmitters released by depolarized cells, was completely inhibited in the absence of extracellular calcium while the response to carbachol was wholly preserved and that to VIP only partially

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Fig. 1. VIP-induced inositol phosphate accumulation is unaffected by calcium removal and presence of TTX in the incubation medium. Isolated ganglia were labelled with [3H]inositol as described in Table I. Incubations were performed in the presence or absence of Ca 2÷ (0.75 mM). Following a 20 min incubation with 10 mM LiCI and, when present, 1/~M tetrodotoxin (TTX), the ganglia were stimulated with VIP (40/~M), carbachol (1 mM) or K ÷ (50 mM) for 20 min in the appropriate medium. The values represent the means + S.D. of 3 independent determinations.

inhibited. Similarily, TTX did not block the responses to carbachol and VIP. Altogether the above results clearly suggest that at least a large part of the observed effect of VIP on inositol lipid breakdown in rat superior cervical ganglia is a direct one, i.e. mediated by VIP receptors or receptors for peptides structurally related to VIP. ha most of the tissues studied so far including the brain and the superior cervical ganglion, VIP has been reported to stimulate adenylate cyclase 7,23. It was, therefore, of interest to investigate the effect of VIP and other agents on cyclic AMP formation in isolated rat superior cervical ganglia. As shown in Table II noradrenaline produced a very marked (17-fold) increase in ganglionic cyclic AMP content. In sharp contrast, arginine vasopressin and VIP (4/~M) induced a very small but significant effect. Voile and Patterson 23 reported a much larger effect of VIP on the total cyclic AMP content of rat superior cervical ganglia. At present, we have no explanation for this apparent discrepancy between the results of Voile and Patterson and ours. At a concentration of 10 nM, which has been shown to induce a half-maximal increase in cyclic AMP in neuronal cells in culture 24,

366 VIP did not alter the level of cyclic A M P in rat superior cervical ganglia. Although one cannot exclude the presence in the rat superior cervical ganglia of VIP and vasopressin-sensitive adenylate cyclase, the possibility that the moderate effect of these peptides on cyclic A M P production could be a consequence of the observed marked increase in inositol lipid breakdown must also be considered. Indeed, in mesangial cells from rat renal glomeruli, vasopressin receptors of the V1 type mediate a large vasopressin-induced stimulation of inositol lipid breakdown. At high concentration vasopressin also produces a moderate increase in intracellular cyclic A M P content which, in this case, could be attributed to prostaglandins produced in response to vasopressin stimulation 1'22. Anyway, the very small magnitude of the cyclic A M P effect of VIP in the rat superior cervical ganglion is an additional argument favoring the conclusion that the VIP effect on inositol lipid breakdown is a direct one. With the aim to further characterize the VIP receptors involved in its effect on inositol lipid breakdown, we determined the concentration dependency of the response and investigated the effects of other peptides of the VIP family (Fig. 2). The half-maximal response to VIP was obtained at a concentration of 1 pM. This value is about 30 times higher than the corresponding one determined for vasopressin (30 nM, this study and ref. 3). It is also greater than the concentration usually needed to provoke a half-maximal accumulation of cyclic A M P in the central nervous system 24. There is experimental evidence suggesting that several classes of VIP receptors are present in the central nervous system. Thus two classes of VIP binding sites (high and low affinity sites) have been identified, and a biphasic pattern of adenylate cyclase activation by VIP has been observed in neuronal membranes of rat cerebral cortex and neurones in primary culture 6'21. The As0 value for VIP-induced inositol lipid breakdown in rat superior cervical ganglia is close to the dissociation constant measured for VIP binding to the low affinity sites. It is also close to the A50 value of 1 p M for Tyr(OH)ase activation by VIP 14. Therefore, the possibility must be considered that, in rat superior cervical ganglia, only low affinity VIP receptors are present and that these receptors are functionally coupled to the transduction mechanism involved in receptor-mediated inositol lipid

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Fig. 2. Concentration-dependent effect of VIP and peptides of the VIP family on inositol lipid breakdown by rat superior cervical ganglia. Ganglia were exposed to increasing concentrations of the indicated peptide as described in the legend to Table I. Results are means + S.D. of 3 determinations.

breakdown. None of the peptides of the VIP family which were tested: secretin, glucagon, PHI and rh G R F were able to stimulate inositol lipid breakdown (Fig. 2) or to accumulate cyclic A M P (Table II). Thus the VIP effect on the rat cervical superior ganglion does not appear to be mediated by a high affinity receptor for a peptide of the VIP family as this was found to be the case for VIP-induced cyclic A M P accumulation in anterior pituitary cells 5. TABLE II Effects of noradrenaline and peptides of the VIP family on the cyclic A M P content of rat superior cervical ganglia

Values are the means _+S.D. for the number of determinations indicated in brackets. Secretin was provided by Dr. Wunsch (Martinsried), glucagon was purchased from Novo Industries, and PHI from Bachem (Switzerland). Addition

cAMP content (% conversion)

None Noradrenaline (0.1 mM) Arginine vasopressin (10/~M) VIP (10 nM) VIP (4pM) Secretin (10 pM) Glucagon (4.6pM) PHI (10#M) rhGRF (4~M)

0.64 _+0.16 (16) 10.93 + 2.89 (8) 1.18 __+0.16 (3) 0.59 + 0.06 (3) 1.46 + 0.28 (3) 0.61 (2) 0.53 (2) 0.62 (2) 0.59 +_0.03 (3)

367 In conclusion, the present study d e m o n s t r a t e s for the first time that V I P can directly stimulate inositol lipid b r e a k d o w n in isolated rat superior cervical ganglia. The receptors responsible for this effect m a y r e p r e s e n t a new class of low affinity V I P receptors; indeed, V I P has been identified in the rat superior cervical ganglia it. They also could be receptors for an, as yet unidentified peptide. The presence of such a peptide was recently p r o p o s e d 4.

This research was s u p p o r t e d by grants from the Minist~re de l'Industrie et de la R e c h e r c h e , Institut National de la Sant6 et de la R e c h e r c h e M6dicale, and Centre National de la Recherche Scientifique. W e thank Dr. M. Manning (Toledo, U . S . A . ) and R. Gaillard ( G e n e v a , Switzerland) for providing the vasopressin antagonist d(CH2)5[Tyr(Et)2,valn,D ArgS]-vasopressin and r h G R F respectively. W e also thank Mich~le Paolucci for secretarial assistance.

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increases tyrosine 3-monooxygenase activity and phospholipid metabolism in the superior cervical ganglion of the rat, J. Pharmacol. Exp. Ther., 229 (1984) 577-582. 13 Horwitz, J., Tsymbalov, S. and Perlman, R.L., Muscarine stimulates the hydrolysis of inositol-containing phospholipids in the superior cervical ganglion, J. Pharmacol. Exp. Ther., 233 (1985) 235-241. 14 Ip, N.Y., Ho, C.K. and Zigmond, R.E., Secretin and vasoactive intestinal peptide acutely increase tyrosine 3-monooxygenase in the rat superior cervical ganglion, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 7566-7569. 15 Jan, Y.N., Jan, L.Y. and Kuffler, S.W., A peptide as a possible transmitter in sympathetic ganglia of the frog, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 1501-1505. 16 Mo, N. and Dun, N.J., Vasoactive intestinal polypeptide facilitates muscarinic transmission in mammalian sympathetic ganglia, Neurosci. Lett., 52 (1984) 19-23. 17 Lundberg, J.M., Terenius, L., H6kfelt, T., Martling, C.R., Tatemoto, K., Mutt, V., Polak, J., Bloom, S. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Scand., 116 (1982) 477-480. 18 MicheU, B., Inositol phosphates: profusion and confusion, Nature (London), 319 (1986) 176-177. 19 North, R.A., Katayama, Y. and Williams, J.T., On the site and mechanism of action of enkephalin on myenteric neurons, Brain Research, 165 (1979) 67-77. 20 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem., 78 (1974) 541-548. 21 Staun-Olsen, P., Ottesen, B., Bartels, P.D., Nielsen, M.H., Gammeltoft, S. and Fahrenkrug, J., Receptors for vasoactive intestinal polypeptide on isolated synaptosomes from rat cerebral cortex. Heterogeneity of binding and desensitization of receptors, J. Neurochem., 39 (1982) 1242-1251. 22 Troyer, D.A., Kreisberg, J.I., Schwertz, D.W. and Vankatachalam, M.A., Effects of vasopressin on phosphoinositides and prostaglandin production in cultured mesangial cells, Am. J. Physiol., 249 (1985) F139-F147. 23 Voile, R.L. and Patterson, B.A., Regulation of cyclic AMP accumulation in a rat sympathetic ganglion: effects of vasoactive intestinal polypeptide, J. Neurochem., 39 (1982) 1195-1197. 24 Weiss, S., Sebben, M., Garcia-Sainz, A. and Bockaert, J., D2-dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture, Mol. Pharmacol., 27 (1985) 595-599.