520
BIOCHEMICAL SOCIETY TRANSACTIONS
could effectively replace the endogenous lipids of other biological membranes. Sarcoplasmicreticulum is unusual in having a highly specialized function requiring only a simple membrane protein composition. In other biological membranes, where there are large numbers of different membrane proteins, lipid heterogeneity may serve at least in part to satisfy their widely different lipid requirements. Robles, E. C. &:Van den Berg, D. (1969) Biochim. Biophys. Acra 187,520-526 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. ( 1 9 7 4 ~Proc. ) Nutl. Acad. Sci. U S A . 71, 622-626 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. (19746) FEBSLert. 41,122-124 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. (1974~) Biochemistry 13,5501-5506 Warren, G. B., Bennett, J. P., Houslay, M. D., Hesketh, T. R., Smith, G. A., & Metcalfe, J. C. (1975)Proc. FEBS Meet. loth, 3-15 Yang, S. F., Freer, S. & Benson, A. A. (1967) J. Biol. Chem. 242,477-484
The Role of Prostaglandin El as an Intercellular Regulator or Modulator of Adenylate Cyclases VL'ITORIO TOMASI,* AGOSTINO TREVISANI, CARLA BIONDI, ANTONIO CAPUZZO and VIRGIL10 PERM Institute of General Physiology, University of Ferrara, 44100 Ferrara, Italy
The hypothesis put forward by Greengard and co-workers (Greengard et al., 1972; Greengard & Kebabian, 1974) on the role of dopamine (3,4-dihydroxyphenethylamine) and cyclic AMP in the modulation of synaptic transmission in the superior cervical ganglion, and the observation (Davis et al., 1971) that pre-ganglionic stimulation at physiological frequencies results in the release of prostaglandins, mainly of the E type, prompted us to investigate the possible functional correlations between dopamine, prostaglandin and cyclic AMP in sympathetic ganglia. When slices of bovine cervical ganglion were incubated in the presence of appropriate amounts of dopamine, noradrenaline, PGE,? and PGF,,, cyclic AMP concentrations were significantly increased in a dose-dependent fashion. Combinations of dopamine and PGEl raised cyclic AMP concentration more than if their effects had been simply additive. Combinations of PGE, with other catecholamines or of dopamine with other prostaglandins produced effects that were simply additive. The synergism of dopamine plus PGEl does not involve action on cyclic AMP phosphodiesterase (EC 3.1.4.17). The a-blocker phentolamine, as well as trillupromazine and fluphenazine, which are known to be potent dopamine antagonists, in the striatum inhibited the response to dopamine without markedly affecting the response to PGEl. These drugs, however, did not impair dopamine plus PGEi synergistic action. These experiments, in our opinion, rule out the existence of an adenylate cyclase (EC 4.6.1.1) responsive to PGEl and modulated by dopamine. In effect the survival of a dopamine plus PGEl synergism, in the presence of appropriate doses of a dopamine-receptor blocker, could hardly be explained on the basis of the latter possibility. These results may be better accounted for by the presence of a dopamine-sensitiveadenylate cyclase modulated by PGEl in the ganglion. In this case, PGEl would be able to increase the number of binding sites for dopamine, thus overcoming the inhibitory action of the blocking drugs. Also the results reported in Fig. 1 are in accordance with 8 modulatory role of PGEl on the dopamine receptor. It can be observed that the extent
* Present address: Institute of General Physiology, University of Napoli, Napoli, Italy. t Abbreviations: PGEl and PGFl., prostaglandin El and Fla. 1977
521
2nd NATIONAL CONGRESS. ITALY 700 -
600 -
500
-
400
-
300
-
n
.-8 3 4-8
.-R Y
m
x
v
3
.-0
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I
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100
-
L
x a
na
Fig. 1. Synergistic control of cyclic AMP concentration by dopamine and PGEl in slices of bovine superior cervical ganglion Slices (40-60mg wet weight) were preincubated for 45min at 37OC in 0.7ml of oxygenated Krebs-Henseleit solution (pH7.4). After the addition of 5m-aminophylline and O ~ ~ M - G Tthe P ,tubes were incubated for 15min; 0.1 mM-EGTA, 0.1 m ~ dopamine and PGEl (0.3 or 15 pg/ml) were then added and the tubes were incubated for Smin, the h a 1 volume being 1.0ml. Reactions were blocked by homogenizing slices in cold 6 % trichloroacetic acid. Supernatants were extracted with aqueous ethyl ether, and cyclicAMP was assayed by the method of Brown et al. (1972). Results are means of four separate experiments run in duplicate. Verticals on top of the bars represent S.E.M. Basal cyclicAMP concentration was 29.5 f3.0pmol/mg of protein. All stimulations were significantly different from control.
of the synergism is correlated with the dose of PGEl added to the slices. Since PGEl alone causes a slight but consistent increase in cyclic AMP concentration, unaffected by dopamine antagonists, the presence of a separate adenylate cyclase merely sensitive to PGEl in the ganglion has to be postulated. It has been reported that carbachol (carbamoylcholine) is able to increase cyclic AMP of rabbit superior cervical ganglion, causing the release of dopamine from the ganglionic interneurons (S.I.F. cells); dopamine in turn directly activates adenylate cyclase in the ganglionic neurons (Greengard & Kebabian, 1974). We observed that 0.1 mM-carbachol slightly but significantly increased cyclic AMP concentration in bovine ganglion slices and that its effect was blocked, as expected,
Vol. 5
522
BIOCHEMICAL SOCIETY TRANSACTIONS
-Blocked by atropine
fi
choiiiiaryic
.:...
=?
Blocked by a-adrenergic
I
Dopamine
I
I
!-
I
0
- 1
I
I
I I
L
(b)
I I
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - A.- - - - - - - - - - -
Fig. 2. Model of theproposed mechanism by which PGE, modulates synaptic transmission in the bovine superior cervical ganglion The model is based on studies reviewed by Libet (1970), Greengard & Kebabian (1974) and Hedqvist (1973). In the lower part of the scheme, within broken lines, the modulatory role of PGEi on post-synaptically located dopamine-sensitive adenylate cyclase, is depicted. by atropine, confirming the muscarinic nature of the receptor. The association of carbachol and PGE, showed an additive effect, and carbachol plus dopamine showed a synergistic one (V. Tomasi, C. Biondi, A. Trevisani, M. Martini & V. Perri, unpublished work). These results suggest that carbachol action on cyclic AMP may be explained not only on the basis of dopamine but also of PGEl release. Incubation of ganglion slices in the presence of labelled arachidonicacid results in aremarkable biosynthesisof labelled prostaglandin (V. Tomasi, C. Biondi, A. Trevisani, M. Martini & V. Perri, unpublished work). Since adenylate cyclase in the nervous system is assumed to be localized mainly on synaptic structures (De Robertis et al., 1967), a set of experiments was carried out on the synaptosomal fraction isolated from the ganglia. Several procedures were tested, including the method of Wilson & Cooper (1972) designed for bovine ganglia, but so far with limited success. We have been able to prepare synaptosomes containing an adenylate cyclase having a basal activity three to four times as high as the homogenate, stimulated by PGEl to an extent comparable with that observed on slices, but with a very poor sensitivity to dopamine. This fraction, moreover, did not show any modulation by PGEl of the dopamine effect. On the basis of electron microscopy and markerenzyme assay, synaptosomal preparations appeared to contain very few, if any, post-synaptic membranes. This would explain the absence of a dopamine-sensitive adenylate cyclase, localized in post-synaptic structures of bovine ganglia (Kebabian et al., 1975). On the other hand the remarkable sensitivity 1977
2nd NATIONAL CONGRESS, ITALY
523
of synaptosomes to PGEl would be consistent with the presence of a PGEl-sensitive adenylate cyclase in pre-ganglionic fibres. In our opinion the data presented here, as well as other unpublished work (V. Tomasi, C. Biondi, A Trevisani, M. Martini & V. Perri), fit into the working hypothesis diagramatically presented in Fig. 2. The stimulation of pre-ganglionic cholinergic fibres involves the activation of muscarinic receptors located on the membrane of interneurons containing dopamine and/or PGE1. The PGEl released from the interneurons regulates ganglionic transmission by acting at two separated sites: (i) in the ganglion neurons as a modulator of the dopamine receptor (dopaminesensitive adenylate cyclase); (ii) in the pre-synaptic nerve terminals by stimulating a PGE,-sensitive adenylate cyclase. The most speculative point of this scheme is the possible existence of interneurons capable of synthesizing and releasing prostaglandins, which in turn would act on different target cells. In this connexion, it may be pointed out that evidence has been given that in the liver PGEl interacts with receptors located on the outer side of the hepatocyte membrane, activating a specific adenylate cyclase (Tomasi & Ferretti, 1975; Tomasi, 1976). It has been shown in addition that the liver cellular population responsive to PGEl is different from the one metabolizing prostaglandins (Ferretti et al., 1976). Brown, B. L., Ekins, R. P. & Albano, J. D. M. (1972) Ado. Cyclic Nucleotide Res. 2 , 2 5 4 0 Davis, H. A., Horton, E. W., Jones, K. B. & Quilliam, J. P. (1971) Br. J. Pharmacol. 42,569-583 De Robertis, E., De Lores Arnaiz, G. R., Alberici, M., Butcher, R. W. & Sutherland, E. W. (1967) J. Biol. Chem. 242,3487-3493
Ferretti, E., Biondi, C . & Tomasi, V. (1976) FEBS Lett. in the press Greengard, P. & Kebabian, J. W. (1974) Fed. Proc. Fed. Am. SOC.Exp. Biol. 33,1059-1067 Greengard, P., McAfee, D. A. & Kebabian, J. W. (1972) Ado. Cyclic Nucleotide Res. 1,337-355 Hedqvist, P. (1973) in Prostaglandins (Ramwell, P. W., ed.), vol. 1, pp. 101-131, Plenum Press, New York and London Kebabian, J. W., Bloom, F. E., Steiner, A. L. & Greengard, P. (1975) Science 190,157-159 Libet, B. (1970) Fed. Proc. Fed. Am. SOC. Exp. Biol. 29, 1945-1956 Tomasi, V. (1976) Immunochemistry of the Cell Membrane, North-Holland, Amsterdam, in the press Tomasi, V. & Ferretti, E. (1975) Mol. Cell. Endocrinol. 2, 221-232 Wilson, W. S. & Cooper, J. R. (1972) J. Neurochem. 19,2779-2790
Structure-Function Relationship of Intestinal and Renal Brush-Border Membrane-Bound Aminopeptidases and Maltases SUZANNE MAROUX, DANIEL LOUVARD, CHRISTIAN VANNIER and MICHEL SfiMgRIVA C.N.R.S., Centre de Biochimie et de Biologie Mol&culaire, 31 Chemin Joseph-Aiguier, 13274 Marseille Cedex 2, France
The membranes of the intestinal and renal brush borders are highly specialized in a typical function of plasma membranes: the active transport of a number of metabolites. These membranes are known to contain a variety of hydrolases (aminopeptidase, disaccharidases, alkaline phosphatase and y-glutamyltransferase). In the case of gut, these hydrolases are involved in the last step of intraluminar digestion. It has been postulated (Ugolev, 1972; Maroux et al., 1973; Malathi et al., 1973) that they also play a role in the transport of digested products across the membrane. The determination of the position occupied by these enzymes with respect to the lipid bilayer is of fundamental importance for a better understanding of their function.
Vol. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
could effectively replace the endogenous lipids of other biological membranes. Sarcoplasmicreticulum is unusual in having a highly specialized function requiring only a simple membrane protein composition. In other biological membranes, where there are large numbers of different membrane proteins, lipid heterogeneity may serve at least in part to satisfy their widely different lipid requirements. Robles, E. C. &:Van den Berg, D. (1969) Biochim. Biophys. Acra 187,520-526 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. ( 1 9 7 4 ~Proc. ) Nutl. Acad. Sci. U S A . 71, 622-626 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. (19746) FEBSLert. 41,122-124 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. (1974~) Biochemistry 13,5501-5506 Warren, G. B., Bennett, J. P., Houslay, M. D., Hesketh, T. R., Smith, G. A., & Metcalfe, J. C. (1975)Proc. FEBS Meet. loth, 3-15 Yang, S. F., Freer, S. & Benson, A. A. (1967) J. Biol. Chem. 242,477-484
The Role of Prostaglandin El as an Intercellular Regulator or Modulator of Adenylate Cyclases VL'ITORIO TOMASI,* AGOSTINO TREVISANI, CARLA BIONDI, ANTONIO CAPUZZO and VIRGIL10 PERM Institute of General Physiology, University of Ferrara, 44100 Ferrara, Italy
The hypothesis put forward by Greengard and co-workers (Greengard et al., 1972; Greengard & Kebabian, 1974) on the role of dopamine (3,4-dihydroxyphenethylamine) and cyclic AMP in the modulation of synaptic transmission in the superior cervical ganglion, and the observation (Davis et al., 1971) that pre-ganglionic stimulation at physiological frequencies results in the release of prostaglandins, mainly of the E type, prompted us to investigate the possible functional correlations between dopamine, prostaglandin and cyclic AMP in sympathetic ganglia. When slices of bovine cervical ganglion were incubated in the presence of appropriate amounts of dopamine, noradrenaline, PGE,? and PGF,,, cyclic AMP concentrations were significantly increased in a dose-dependent fashion. Combinations of dopamine and PGEl raised cyclic AMP concentration more than if their effects had been simply additive. Combinations of PGE, with other catecholamines or of dopamine with other prostaglandins produced effects that were simply additive. The synergism of dopamine plus PGEl does not involve action on cyclic AMP phosphodiesterase (EC 3.1.4.17). The a-blocker phentolamine, as well as trillupromazine and fluphenazine, which are known to be potent dopamine antagonists, in the striatum inhibited the response to dopamine without markedly affecting the response to PGEl. These drugs, however, did not impair dopamine plus PGEi synergistic action. These experiments, in our opinion, rule out the existence of an adenylate cyclase (EC 4.6.1.1) responsive to PGEl and modulated by dopamine. In effect the survival of a dopamine plus PGEl synergism, in the presence of appropriate doses of a dopamine-receptor blocker, could hardly be explained on the basis of the latter possibility. These results may be better accounted for by the presence of a dopamine-sensitiveadenylate cyclase modulated by PGEl in the ganglion. In this case, PGEl would be able to increase the number of binding sites for dopamine, thus overcoming the inhibitory action of the blocking drugs. Also the results reported in Fig. 1 are in accordance with 8 modulatory role of PGEl on the dopamine receptor. It can be observed that the extent
* Present address: Institute of General Physiology, University of Napoli, Napoli, Italy. t Abbreviations: PGEl and PGFl., prostaglandin El and Fla. 1977
521
2nd NATIONAL CONGRESS. ITALY 700 -
600 -
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400
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300
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n
.-8 3 4-8
.-R Y
m
x
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.-0
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100
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L
x a
na
Fig. 1. Synergistic control of cyclic AMP concentration by dopamine and PGEl in slices of bovine superior cervical ganglion Slices (40-60mg wet weight) were preincubated for 45min at 37OC in 0.7ml of oxygenated Krebs-Henseleit solution (pH7.4). After the addition of 5m-aminophylline and O ~ ~ M - G Tthe P ,tubes were incubated for 15min; 0.1 mM-EGTA, 0.1 m ~ dopamine and PGEl (0.3 or 15 pg/ml) were then added and the tubes were incubated for Smin, the h a 1 volume being 1.0ml. Reactions were blocked by homogenizing slices in cold 6 % trichloroacetic acid. Supernatants were extracted with aqueous ethyl ether, and cyclicAMP was assayed by the method of Brown et al. (1972). Results are means of four separate experiments run in duplicate. Verticals on top of the bars represent S.E.M. Basal cyclicAMP concentration was 29.5 f3.0pmol/mg of protein. All stimulations were significantly different from control.
of the synergism is correlated with the dose of PGEl added to the slices. Since PGEl alone causes a slight but consistent increase in cyclic AMP concentration, unaffected by dopamine antagonists, the presence of a separate adenylate cyclase merely sensitive to PGEl in the ganglion has to be postulated. It has been reported that carbachol (carbamoylcholine) is able to increase cyclic AMP of rabbit superior cervical ganglion, causing the release of dopamine from the ganglionic interneurons (S.I.F. cells); dopamine in turn directly activates adenylate cyclase in the ganglionic neurons (Greengard & Kebabian, 1974). We observed that 0.1 mM-carbachol slightly but significantly increased cyclic AMP concentration in bovine ganglion slices and that its effect was blocked, as expected,
Vol. 5
522
BIOCHEMICAL SOCIETY TRANSACTIONS
-Blocked by atropine
fi
choiiiiaryic
.:...
=?
Blocked by a-adrenergic
I
Dopamine
I
I
!-
I
0
- 1
I
I
I I
L
(b)
I I
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - A.- - - - - - - - - - -
Fig. 2. Model of theproposed mechanism by which PGE, modulates synaptic transmission in the bovine superior cervical ganglion The model is based on studies reviewed by Libet (1970), Greengard & Kebabian (1974) and Hedqvist (1973). In the lower part of the scheme, within broken lines, the modulatory role of PGEi on post-synaptically located dopamine-sensitive adenylate cyclase, is depicted. by atropine, confirming the muscarinic nature of the receptor. The association of carbachol and PGE, showed an additive effect, and carbachol plus dopamine showed a synergistic one (V. Tomasi, C. Biondi, A. Trevisani, M. Martini & V. Perri, unpublished work). These results suggest that carbachol action on cyclic AMP may be explained not only on the basis of dopamine but also of PGEl release. Incubation of ganglion slices in the presence of labelled arachidonicacid results in aremarkable biosynthesisof labelled prostaglandin (V. Tomasi, C. Biondi, A. Trevisani, M. Martini & V. Perri, unpublished work). Since adenylate cyclase in the nervous system is assumed to be localized mainly on synaptic structures (De Robertis et al., 1967), a set of experiments was carried out on the synaptosomal fraction isolated from the ganglia. Several procedures were tested, including the method of Wilson & Cooper (1972) designed for bovine ganglia, but so far with limited success. We have been able to prepare synaptosomes containing an adenylate cyclase having a basal activity three to four times as high as the homogenate, stimulated by PGEl to an extent comparable with that observed on slices, but with a very poor sensitivity to dopamine. This fraction, moreover, did not show any modulation by PGEl of the dopamine effect. On the basis of electron microscopy and markerenzyme assay, synaptosomal preparations appeared to contain very few, if any, post-synaptic membranes. This would explain the absence of a dopamine-sensitive adenylate cyclase, localized in post-synaptic structures of bovine ganglia (Kebabian et al., 1975). On the other hand the remarkable sensitivity 1977
2nd NATIONAL CONGRESS, ITALY
523
of synaptosomes to PGEl would be consistent with the presence of a PGEl-sensitive adenylate cyclase in pre-ganglionic fibres. In our opinion the data presented here, as well as other unpublished work (V. Tomasi, C. Biondi, A Trevisani, M. Martini & V. Perri), fit into the working hypothesis diagramatically presented in Fig. 2. The stimulation of pre-ganglionic cholinergic fibres involves the activation of muscarinic receptors located on the membrane of interneurons containing dopamine and/or PGE1. The PGEl released from the interneurons regulates ganglionic transmission by acting at two separated sites: (i) in the ganglion neurons as a modulator of the dopamine receptor (dopaminesensitive adenylate cyclase); (ii) in the pre-synaptic nerve terminals by stimulating a PGE,-sensitive adenylate cyclase. The most speculative point of this scheme is the possible existence of interneurons capable of synthesizing and releasing prostaglandins, which in turn would act on different target cells. In this connexion, it may be pointed out that evidence has been given that in the liver PGEl interacts with receptors located on the outer side of the hepatocyte membrane, activating a specific adenylate cyclase (Tomasi & Ferretti, 1975; Tomasi, 1976). It has been shown in addition that the liver cellular population responsive to PGEl is different from the one metabolizing prostaglandins (Ferretti et al., 1976). Brown, B. L., Ekins, R. P. & Albano, J. D. M. (1972) Ado. Cyclic Nucleotide Res. 2 , 2 5 4 0 Davis, H. A., Horton, E. W., Jones, K. B. & Quilliam, J. P. (1971) Br. J. Pharmacol. 42,569-583 De Robertis, E., De Lores Arnaiz, G. R., Alberici, M., Butcher, R. W. & Sutherland, E. W. (1967) J. Biol. Chem. 242,3487-3493
Ferretti, E., Biondi, C . & Tomasi, V. (1976) FEBS Lett. in the press Greengard, P. & Kebabian, J. W. (1974) Fed. Proc. Fed. Am. SOC.Exp. Biol. 33,1059-1067 Greengard, P., McAfee, D. A. & Kebabian, J. W. (1972) Ado. Cyclic Nucleotide Res. 1,337-355 Hedqvist, P. (1973) in Prostaglandins (Ramwell, P. W., ed.), vol. 1, pp. 101-131, Plenum Press, New York and London Kebabian, J. W., Bloom, F. E., Steiner, A. L. & Greengard, P. (1975) Science 190,157-159 Libet, B. (1970) Fed. Proc. Fed. Am. SOC. Exp. Biol. 29, 1945-1956 Tomasi, V. (1976) Immunochemistry of the Cell Membrane, North-Holland, Amsterdam, in the press Tomasi, V. & Ferretti, E. (1975) Mol. Cell. Endocrinol. 2, 221-232 Wilson, W. S. & Cooper, J. R. (1972) J. Neurochem. 19,2779-2790
Structure-Function Relationship of Intestinal and Renal Brush-Border Membrane-Bound Aminopeptidases and Maltases SUZANNE MAROUX, DANIEL LOUVARD, CHRISTIAN VANNIER and MICHEL SfiMgRIVA C.N.R.S., Centre de Biochimie et de Biologie Mol&culaire, 31 Chemin Joseph-Aiguier, 13274 Marseille Cedex 2, France
The membranes of the intestinal and renal brush borders are highly specialized in a typical function of plasma membranes: the active transport of a number of metabolites. These membranes are known to contain a variety of hydrolases (aminopeptidase, disaccharidases, alkaline phosphatase and y-glutamyltransferase). In the case of gut, these hydrolases are involved in the last step of intraluminar digestion. It has been postulated (Ugolev, 1972; Maroux et al., 1973; Malathi et al., 1973) that they also play a role in the transport of digested products across the membrane. The determination of the position occupied by these enzymes with respect to the lipid bilayer is of fundamental importance for a better understanding of their function.
Vol. 5
