Regulation by Secretin, Vasoactive Intestinal Peptide

Proc. Natl. Acad. Sci. USA Vol. 77, No. 11, pp. 6907-6911, November 1980 Neurobiology

Regulation by secretin, vasoactive intestinal peptide, and somatostatin of cyclic AMP accumulation in cultured brain cells (glioblasts/peptides/adenylate cyclase/receptors/gastrointestinal hormones) DIETRICH VAN CALKER, MARGARETE MOLLER, AND BERND HAMPRECHT* Max-Planck-Institut fur Biochemie, 8033 Martinsried, Federal Republic of Germany Communicated by Martin Lindauer, August 11, 1980

ABSTRACT Secretin stimulates the accumulation of cyclic Germany. Synthetic VIP (porcine) was from Peninsula Labo- AMP (half maximally stimulating concentration: 10-20 nM) in ratories, San Carlos, CA. Identical results were obtained with cultured mouse brain cells mainly consisting of glioblasts. the two preparations of somatostatin and VIP. The sources of Vasoactive intestinal peptide (VIP) is much less ptent in raising the level of cyclic AMP in these cultures. The effect of secretin all other materials are given elsewhere (7, 8). but not that of VIP is inhibited by secretin45-27), a synthetic Cell Culture. Brain cells were obtained from neonatal antagonist of secretin. Stimulation of the adrenergic a-receptors BALB/c mice by mechanical dissociation (7). A suspension of and the adenosine A1-receptors present on the cells attenuates 3 X 106 viable cells (viability 50%, as determined by exclusion the increase in cyclic AMP evoked by secretin and VIP. Soma- of nigrosin) in 5 ml of growth medium (90% Dulbecco's mod- tostatin at low concentrations inhibits the accumulation of cy- ified Eagle's medium, 10% fetal calf serum, Na penicillin at 20 clic AMP (half-maximally inhibitory concentration: 3 nM), in sulfate at 20 the absence or presence of secretin, VIP, or isoproterenol. The units/ml, streptomycin lug/ml) was seeded onto results suggest that secretin might regulate the concentration replica plastic petri dishes 60 mm in diameter and cultured at of cyclic AMP in brain and provoke the question of a possible 370C in a humidified atmosphere of 90% air/10% CO2 (pH of involvement of glial cells in the action of peptide hormones in medium: 7.4 at 370G). The medium was renewed once on the the brain. sixth day of culture irrespective of the total length of the culture period (see legends of figures and table). The pH of the medium Various peptides are known to be common to the brain and the does not change up to a culture time of 4 weeks. Such cultures gastrointestinal tract and pancreas (1). Some of these peptides consist mainly of epithelioid cells (9) considered to be astroblasts, (e.g., substance P, somatostatin, enkephalin) have been shown which can differentiate to resemble mature astrocytes (10, 11). to elicit specific behavioral changes or changes in specific They are employed as models for glial cells (5, 7, 12, 13), be- neuron firing rates or patterns (for review see refs. 2-4). They cause they express presumptive glial markers (5, 14-17) (for are, therefore, hypothesized to act on neurons as neuromodu- review see ref. 5). To ensure that the cells we used closely re- lators or neurotransmitters. Some neurohormones are also semble the rat brain cells described by others (5, 9-12, 14) we known to act on glial cells by regulating the intracellular level have confirmed (unpublished data) the following properties: of cyclic AMP (for review see ref. 5). The possibility had to be (i) morphology (9, 10), (Ui) morphological differentiation to considered that this would not only hold for biogenic amines astrocyte-like cells in the presence of butyryl derivatives of such as norepinephrine but also for peptide hormones. There- cyclic AMP (10, 11), (iii) presence of the glial fibrillary acidic fore, we investigated the effects of peptide hormones on the protein in the majority of cells present in the cultures as shown accumulation of cyclic AMP in glial cell cultures derived from by immunofluorescence (14). As already reported for rat brain neonatal mouse brain. We report-here that somatostatin inhibits cells (9), no morphologically distinguishable neurons can be the basal and the hormone-induced accumulation of cyclic observed in the cultures. In addition, the absence from such AMP in such primary cultures. Furthermore, we report that cultures of the neuron-specific antigens 14-3-2, DI, D2, and D3 the gastrointestinal hormones secretin and vasoactive intestinal (17) and of the neuronal marker enzyme glutamate decarbox- peptide (VIP) stimulate the accumulation of cyclic AMP in ylase (18, 19) indicates that neurons are missing from such these cultures, probably via different types of receptors. Thus cultures. a possible role of peptide hormones in the function of glial cells Experimental Incubation. A detailed description of the is indicated. In addition, the results suggest that secretin or a incubation procedure is given elsewhere (20). Briefly, before closely related compound might exert regulatory influence in the experimental incubation the cultures were washed with 2 the brain. A preliminary report of some of these results has been ml of incubation medium (identical with Dulbecco's modified presented at a conference (6). Eagle's medium except that it contained 24.6 g of NaHCO3 per liter instead of 37 g and that it was adjusted to 320 milliosmoles MATERIALS AND METHODS per liter by addition of NaCl). Thereafter they were incubated Materials. Dulbecco's modified Eagle's medium and fetal (10 min, 37°C, pH 7.4) in 2 ml of incubation medium con- bovine serum (GIBCO) were from C. Roth, Karlsruhe, Federal taining the various additions, before the concentration of in- Republic of Germany. A partially purified preparation of VIP tracellular cyclic AMP was determined (6, 7, 21). The number (porcine) was kindly donated by V. Mutt, Stockholm, Sweden. of cells per plate at the time of incubation cannot be given, Isolated somatostatin (cyclic; bovine) was from Beckman, because it has not been possible to completely detach the cells Munchen, Federal Republic of Germany. Synthetic somatos- and generate a suspension of single cells without destroying a tatin (cyclic; bovine) and secretin (porcine) were kind gifts of Abbreviations: iBuMeXan, isobutylmethylxanthine; VIP, vasoactive L. Moroder and E. Wuinsch, Martinsried, Federal Republic of intestinal peptide; Ro 20-1724, 4-(3-butoxy-4-methoxybenzyl)-2- imidazolidinone; secretin-(5-27), fragment of secretin consisting of The publication costs of this article were defrayed in part by page residues 5-27. charge payment. This article must therefore be hereby marked "ad- * To whom reprint requests should be addressed. Present address: vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Physiologisch-Chemisches Institut der Universitit, Koellikerstrasse this fact. 2, 8700 Wuirzburg, Federal Republic of Germany. 6907 Downloaded by guest on October 2, 2021 6908 Neurobiology: van Calker et al. Proc. Natl. Acad. Sci. USA 77 (1980) large number of them. The results are means + standard de- viations of triplicate incubations carried out in parallel on replica plates. Each experiment has been carried out at least twice (with comparable results). RESULTS Secretin and VIP. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (iBuMeXan; ref. 22), secretin and VIP increase the content of cyclic AMP in the cultured brain cells (Fig. 1, curve a; Fig. 2, curves a and c). This increase cannot be due to an action of the peptides at the receptors already known to be present on the cells-i.e., adrenergic a- and f-receptors (7) and adenosine Al- and A2-receptors (13, 21, 23)-because the effects are not prevented by the corre- sponding antagonists phentolamine, propranolol, and iBu- MeXan, respectively (Table 1). The phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724; ref. 24) was used in experiments 1 and 4 (Table 1), because iBuMeXan is also a potent antagonist of adenosine Al- and A2-receptors (21, 23). However, 3 ,M secretin-(5-27), a synthetic fragment of secretin known as an antagonist of secretin cc 9 8 7 6 (25), produces a parallel shift to the right in the dose-response -log[peptide] (M) curve for secretin (Fig. 1, curve b). In contrast, 3 AM secretin- FIG. 2. Stimulation by various concentrations ofsecretin (curves (5-27) does not antagonize the effect of VIP (Fig. 2, curves c a and b) and VIP (synthetic; curves c and d) of cyclic AMP accumu- and d). VIP is much less potent than secretin in stimulating the lation in cultured brain cells in the absence (curves a and c) or pres- accumulation of cyclic AMP in the cultures (Fig. 2). Fig. 2, ence (curves b and d) of 3MuM secretin-(5-27). Cells were cultured for 28 days. All incubations were in the presence of 0.5 mM iBuMeXan. curves c and d, might suggest a biphasic response of the cultures Accumulations were, in terms of pmol of cyclic AMP per mg of pro- to VIP. However, in two repetitions of the experiment no bi- tein: control, 44 + 4; 0.1 AM secretin, 3700 ± 90; 0.1 MM VIP, 800 + phasic dose-response curves were obtained. From one cell batch 140; secretin + VIP, 4660 I 150. Protein, 1.2 mg per plate. Other de- to another, at a given hormone concentration, the responses to tails as in Fig. 1. secretin and VIP vary appreciably. Such as yet unexplained variability of the response has also been observed for isoprote- The increase in the level of cyclic AMP evoked by secretin renol in the brain cell system and for other hormones in per- and VIP is completed within, respectively, 5 or 2 min (Fig. 3, manent cell lines (7). curves a and b). Secretin and VIP increase the level of cyclic AMP also in the absence of a phosphodiesterase inhibitor, although the effect is about one order of magnitude lower (Fig. 3, curve c, and legend of Fig. 3). The stimulation by secretin is probably a direct effect of the peptide on adenylate cyclase 5000 activity and not due to the secretin-induced release of a stimulatory factor. This was concluded from the following experiment (data not shown): Cells were incubated with 10 nM secretin (10 min, 370C). The medium was removed and supple- u4000 a mented with 3 MM secretin-(5-27). Cultures that were incu- 0a bated with this medium showed no substantial increase in the level of cyclic AMP, while controls [no secretin-(5-27) added] revealed the expected increase. o .3000I The cultured brain cells express adrenergic a-receptors and adenosine Al-receptors, the stimulation of which causes an inhibition of a 13-adrenoceptor-mediated increase in the level of cyclic AMP (refs. 7 and 21). Similarly, the stimulation by Q 2000 secretin and VIP of cyclic AMP accumulation is attenuated by norepinephrine (an a- and f3-adrenergic agonist) in the presence of the f3-adrenergic antagonist propranolol (Table 1). The inhibition is blocked by the a-adrenergic antagonist phento- 1000 lamine. Al-agonists also prevent the increase in the level of cyclic AMP caused by stimulatory hormones, as exemplified by N6-(phenylisopropyl)adenosine in the case of VIP and by adenosine in the case of secretin (Table 1). These effects are blocked by the Al-antagonists iBuMeXan (Table 1) and theo- 9 8 7 6 phylline (not shown). -log[secretin ] (M) Somatostatin. The increase in cyclic AMP caused by secretin FIG. 1. Stimulation by secretin of cyclic AMP accumulation in and VIP is inhibited by somatostatin in a dose-dependent cultured brain cells in the absence (curve a) or presence (curve b) of manner (Fig. 4). Also, the stimulation by isoproterenol of cyclic 3 AM secretin-(5-27). Brain cells from neonatal mice were cultured for 20 days. All incubations were in the presence of 0.5 mM iBu- AMP accumulation (7) (Fig. 5A) and the basal level of cyclic MeXan; with no further addition there was 35 i 3 pmol ofcyclic AMP AMP that is observed in the presence of iBuMeXan (Fig. 5B) per mg of protein. Protein content was 0.9 mg per plate (60 mm in are decreased by somatostatin. Half-maximal inhibition is found diameter). Bars indicate SD. at a concentration (ICso) of about 3 nM. It has been hypothe- Downloaded by guest on October 2, 2021 Neurobiology: van Calker et al. Proc. Natl. Acad. Sci. USA 77 (1980) 6909

Table 1. Inhibition by a-adrenergic agonists and adenosine A1-agonists of the increase in the level of cyclic AMP evoked by secretin and VIP Cyclic AMP, pmol/mg Exp.* Addition (1uM) protein *2000 j 1 Control (Ro 20-1724, 500) 6 ± 1 Secretin (0.01) 410 A 25 Secretin + adenosine (10)t 180 I 26 iBuMeXan (500) 15 i 3 0 Secretin + iBuMeXan 1880 1 130 Secretin + iBuMeXan + adenosine 1550 + 230 2 Control (iBuMeXan, 500) 14 + 1 Secretin (0.1) 750 : 150 04 Secretin + propranolol (10)t 990 ± 160 Secretin + phentolamine (10)t 860 A 90 Secretin + propranolol + phentolamine 1000 + 140 1000- Secretin + propranolol + norepinephrine (10) 350 + 60 Secretin + propranolol + phentolamine 1050 + 130 + norepinephrine Propranolol + norepinephrine 140 14 3 Control (iBuMeXan, 500) 30 L 3 VIP (0.1) 1140 I40 Norepinephrine (10) + propranolol (100) 30 + 3 VIP + propranolol 1060 1 90 VIP + propranolol + norepinephrine 410 I 60 VIP + propranolol + phentolamine (100) 730 i 100 Ic VIP + propranolol + phentolamine 770 ± 220 + norepinephrine 2 5 10 4A Control (Ro 20-1724, 500) 10 I 4 Time of incubation, min VIP (0.1) 270 I 45 FIG. 3. Time course of the stimulation by secretin (0.1 uM; curves VIP + N6-(phenylisopropyl)adenosine (0.1) 76 : 16 a and c) and VIP (0.1 AM; synthetic; curve b) of cyclic AMP accu- 4B Control (iBuMeXan, 500) 56 : 19 mulation in cultured brain cells in the presence (curves a and b) or VIP 302 k 70 absence (curve c) of 0.5 mM iBuMeXan. VIP (curve b) increases the VIP + N6-(phenylisopropyl)adenosine 230 ± 46 level of cyclic AMP also in the absence of iBuMeXan. Accumulations were, in terms of pmol of cyclic AMP per mg of protein: control, 10 Cells were cultured for 15 (experiments 1-3) or 30 (experiments 4A + 5; VIP (0.1 MAM), 40 ± 4. Curves a, c: cells were cultured for 17 days; and 4B) days. Protein content, in terms of mg per plate: experiments 1.2 mg of protein per plate. Curve b: cultures are replicas ofthose in 1 and 2, 1.0; experiment 3, 1.5; experiments 4A and 4B, 1.1. Fig. 2. Other details as in Fig. 1. * All incubations ofthese experiments were carried out in the presence of the phosphodiesterase inhibitor indicated in the control. (nM) concentrations (first phase; mediated by high-affinity VIP t Adenosine (10lM), N6-(phenylisopropyl)adenosine, propranolol, receptors) is not at all (25) or only slightly (30) inhibited by se- and phentolamine did not substantially alter the level of cyclic cretin-(5-27). Its action at high (,gM) concentrations [second AMP. phase; mediated by low-affinity VIP receptors that are probably identical with secretin receptors (25)] is effectively inhibited sized that somatostatin acts on opiate receptors (26, 27). How- by the secretin fragment (25). In contrast, secretin-(5-27) does ever, this cannot be true for the cultures investigated here, not inhibit the action of VIP on cultured brain cells. (iii) For because opioids do not inhibit the effect of isoproterenol on the unknown reasons the brain cells are less sensitive to secretin than cultured brain cells (unpublished data) and the opiate antagonist the pancreatic acinar cells are. In addition, their sensitivity to naloxone does not block the effect of somatostatin (Fig. 5A, VIP is far below that of pancreatic acinar cells (25, 30), fat cells curve b). Furthermore, it has been reported that such cultures (31), liver (31, 32), intestinal epithelium (33), pituitary (34), and lack specific binding sites for naloxone (28). Separate receptors brain (35, 36). In these cases the VIP receptors that are highly for opiates and somatostatin have already been demonstrated sensitive to VIP also display a low sensitivity to secretin, as de- for neuroblastoma-glioma hybrid cells (29). The effect of so- rived from receptor binding studies (32, 33, 35, 36) and mea- matostatin on cultured brain cells is probably due to an action surements of adenylate cyclase (31, 34) or of concentrations of at receptors specific for this peptide, because neither A1- nor cellular cyclic AMP (33, 34). a-adrenergic antagonists influence its action (not shown). In the dose-response curve for VIP action on the brain cells a plateau is not reached even at a concentration as high as 1 ,uM. DISCUSSION The insensitivity to the secretin antagonist could be consistent with the view that this curve corresponds to the ascending part Both secretin and VIP stimulate the accumulation of cyclic of the first phase of the VIP dose-response curve in the pan- AMP in cultured mouse brain cells. The action of secretin is creatic acinar cell system but is shifted to the right on the con- inhibited by secretin-(5-27). Essentially the same situation has centration axis. In spite of its low potency, VIP appears to cause been reported for the pancreatic acinar cell system (25, 30). a quite specific effect, because (i) the actions of both VIP and However, three differences between the two systems should be secretin fail to be inhibited by antagonists of receptors already pointed out: (i) In the brain cell system secretin is much more known to be present on the cells; (H) the effect of secretin, but potent than VIP in raising the level of cyclic AMP. In the not of VIP, is suppressed by secretin-(5-27); (iii) various other pancreatic acinar cell system they are of comparable potency (hormonal) peptides such as thyroliberin, luteoliberin, brady- (25). (ii) The dose-response curve for the action of VIP on kinin, glucagon, and insulin do not affect the level of cyclic pancreatic acinar cells is biphasic (25). The action of VIP at low AMP in the cultured brain cells (data not shown). Downloaded by guest on October 2, 2021 6910 Neurobiology: van Calker et al. Proc. Natl. Acad. Sci. USA 77 (1980) curve has been obtained for the activation by VIP of adenylate cyclase in guinea pig brain membranes (37) or rat brain slices (38) that is very similar to that for VIP in the culture system (Fig. 2). A possibly even lower sensitivity to VIP has been reported for adenylate cyclase in rat brain membranes (39). In addition, the stimulations are small (39), 25-50% over basal 0) activity at 10 ,uM VIP in comparison to approximately 3000% i4 I ' at 1 IAM VIP in the brain cell culture system. The degradation of the peptides VIP, secretin, and somatostatin during the standard 10-min incubation period has not been assessed and thus cannot be excluded. In appreciation of the data on hor- 04 mone sensitivities this has to be borne in mind. 6000 The results presented here clearly indicate that the effect of secretin cannot be mediated by receptors specific for VIP but is most likely due to the activation of receptors specific for secretin or a very closely related compound. The effect of VIP is unlikely to be mediated by secretin receptors, because the action of VIP-in contrast to that of secretin-is not antagon- ized by secretin-(5-27). Whether or not the effect of VIP is mediated by receptors specific for it or for a yet-unknown compound remains to be clarified. Notwithstanding this, the most likely explanation of the effects on adenylate cyclase of 9 8 7 6 VIP and secretin is that they are mediated by different recep- -log[somatostatin] (M) tors. The presence of receptors for these peptide hormones and FIG. 4. Inhibition by somatostatin (isolated; curve a) of the in- somatostatin will have to be demonstrated by binding studies crease in the level of cyclic AMP evoked by 0.1 MM secretin (curve a) using radiolabeled peptide hormones. or 0.1 MuM VIP (synthetic; curve b), in cultured mouse brain cells. Somatostatin inhibits the increase in cyclic AMP elicited by Curve a: cells were cultured for 23 days. All incubations were in the presence of0.5 mM iBuMeXan. Accumulations were, in terms ofpmol three hormonal substances: secretin, VIP, and the f3-adrenergic of cyclic AMP per mg of protein: no further addition, 25 + 6; 0.1 uM agonist isoproterenol. The chemical differences, especially those somatostatin, 8 ± 2. Protein content was 1.3 mg per plate. Curve b: between the peptides and the catecholamine, provide indirect cultures are replicas of those of Fig. 2. Other details as in Fig. 1. evidence against a competitive inhibition exerted by somatostatin. The presence of VIP (40-42) and somatostatin (43-45) As reported previously (7), solutions of two commercially in various areas of the brain has been reported. Furthermore, available preparations of VIP proved to be a mixture of dif- there is evidence for adenylate cyclase activity in brain that is ferent polypeptides. Similar results were obtained with a inhibited by somatostatin (46)-albeit only at concentrations preparation of VIP that was isolated by V. Mutt, Stockholm, above 10,M-and stimulated by VIP (37-39). Our finding that Sweden. More recently we have obtained (F. Propst and B. these peptides mediate, respectively, a stimulation and an in- Hamprecht, unpublished) evidence that the major contaminant hibition of cyclic AMP accumulation in cultured brain cells is rapidly formed in an aqueous solution of pure synthetic provides additional evidence for a regulatory function of these peptide (Peninsula Laboratories); it is most likely a somewhat or related peptides on the adenylate cyclase system of certain altered form of VIP. Thus, although we do not hold it likely, brain cells. Because the effect of secretin on the cultured brain at the present time we cannot exclude with certainty that the cells could be clearly distinguished from that of VIP, it is in- effects observed with VIP preparations are caused by a con- ferred that secretin also may have a functional role in the brain. taminant derived from VIP. Indeed, after the experiments reported here had been finished, The low potency of VIP in the brain cell culture system is Mutt et al. (47) reported the extraction of a secretin-like probably not an artefact of the culture system; a dose-response bioactive material from pig brain.

be 6000

-4 FIG. 5. Inhibition by somatostatin (isolated) of cyclic AMP accumulation in the presence (A) or absence (B) of 1 gM isoproterenol. All incubations were in the Q 4000- presence of 0.5 mM iBuMeXan. (A) Curve a, absence of naloxone; curve b, presence of 10 uM naloxone. Cul- tures were replicas of those in Fig. 1; incubation in the 0 presence of naloxone alone: 80 ± 20 pmol of cyclic AMP per mg of protein. (B) Cells were cultured for 23 days; "'-i protein content, 1.2 mg per plate. The content of cyclic AMP in the incubation medium amounts to about 10% of the total, whether or not somatostatin is present 6 (separate experiment, data not shown). Other details -log[somatostatin I (M) as in Figs. 1 and 2. Downloaded by guest on October 2, 2021 Neurobiology: van Calker et al. Proc. Natl. Acad. Sci. USA 77 (1980) 6911

The brain cell cultures consist to a large extent of glial fi- 13. van Calker, D. (1977) Dissertation (Univ. Munich, Federal Re- brillary acid-protein-containing epithelioid cells (9) capable public of Germany). of differentiating into astrocyte-like cells (5, 10-12, 14, 15) and 14. Bock, E., Moller, M., Nissen, C. & Sensenbrenner, M. (1977) FEBS thus may be called astroblasts. Besides these, minor populations Lett. 83, 207-211. of other cells are observed that all together may not account for 15. Lim, R., Turrif, D. E., Troy, S. S., Moore, B. W. & Eng, L. F. (1977) Science 195, 195-196. more than an estimated 10-15% of the total cell population (5). 16. Breen, G. A. M. & de Vellis, J. (1974) Dev. Biol. 41, 255-266. These cells include ependymal cells identified by their beating 17. Bock, E., Jorgensen, 0. S., Dittmann, L. & Eng, L. F. (1975) J. cilia, small cells that are probably oligodendroblasts (48), and Neurochem. 25, 867-870. phagocytic cells. The present findings appear the more inter- 18. Schousboe, A., Svenneby, G. & Hertz, L. (1977) J. Neurochem. esting because the magnitude of the hormonal responses allows 29,999-1005. their assignment (7) only to the majority of cells in the cul- 19. Wu, P. H., Durden, D. A. & Hertz, L. (1979) J. Neurochem. 32, tures-i.e., the cells of glial (astroblast) character (5, 9, 14). Thus 379-390. in the central nervous neurons have been 20. Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1974) in far, system only Proceedings of the 9th International Congress Collegium In- considered and found as targets of peptide hormones (2-4). ternationale Neuropsycho-pharmacologicum, Paris (Excerpta However, the present results suggest that glial cells also can be Medica, Amsterdam), pp. 956-969. regulated by peptide hormones. This provokes the question of 21. van Calker, D., Miller, M. & Hamprecht, B. (1978) Nature the involvement of glial cells in the actions of peptides in the (London) 276,839-841. brain. Interestingly, the peptide hormones acting on the brain 22. Schultz, J. & Hamprecht, B. (1973) Naunyn-Schmiedeberg's cultures also regulate the level of cyclic AMP in neuroblas- Arch. Pharmacol. 278,215-225. toma-glioma hybrid cells. Thus, in the clonal cell line secretin 23. van Calker, D., Muller, M. & Hamprecht, B. (1979) J. Neuro- and VIP increase the of AMP whereas soma- chem. 33, 999-1005. level cyclic (8), 24. Sheppard, H. & Wiggan, C. (1970) Mol. Pharmacol. 7, 111- tostatin inhibits the elevation caused by prostaglandin E1 (29). 115. It should be noted, however, that these hybrids display a series 25. Robberecht, P., Conlon, T. P. & Gardner, J. D. (1976) J. Biol. of neuronal properties with no glial properties detected yet Chem. 251, 4635-4639. (49). 26. Martin, J. B., Audet, J. & Saunders, A. 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