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A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain
Int. J. Biochem. Vol. 19, No. 8, pp. 737-740. 1987
0020-71IX/87 $3.00+ 0.00 Copyright © 1987PergamonJournals Ltd
Printed in Great Britain. All rights reserved
A COMPARISON OF THE EFFECTS OF SUBSTANCE P A N D SHORTER A N A L O G U E S ON THE SYNAPTOSOMAL ATPases ACTIVITIES IN THE RAT BRAIN LILLA LACHOWICZand GRAZYNAJANISZEWSKA II Department of Biochemistry, Institute of Physiology and Biochemistry, School of Medicine, Lindleya 6, 90-131 L6d~, Poland (Received 24 November 1986)
Abstract--The influence in vitro of SP and C-terminal fragments of analogues SP(5_ll) (
INTRODUCTION The endogenous undecapeptide substance P (SP) is a member of a family of peptides known as the tachykinins which possess the conserved carboxyl terminal sequence-Phe-X--Gly-Leu-Met-NH2 (Deutch et al., 1985). More neuronal pathways of action have been demonstrated than for any of the other peptide transmitters. However, the mechanism of the excitatory action of SP on neuronal cells still remains unclear. Substance P has been reported to excite neurons by depolarizing them with a concomitant change in membrane resistance (Krnjevic, 1977; Nicoll, 1978; Zieglg/insberger and Tulloch, 1979). On the other hand, an increase in membrane resistance frequently accompanies substance Pinduced depolarization, an inactivation of the K + conductance, which has been suggested to be involved in the underlying mechanism. Further, a more complex mechanism has been proposed: a simultaneous increase in Na ÷ conductance and a decrease in K + conductance (Dun and Minota, 1981; Minota et al., 1981). It has become evident that some neuropeptides regulate the movement of ions down these concentration gradients, through ion channels in the membrane (Eardly and McGree, 1985). But distinctly less information concerns neuropeptide effects on active
ions transport, and therefore, on ATPases pumps, which are dependent on hydrolysis of ATP. Substitution of amino acid residues with D-amino acids is known to confer antagonistic activity. By replacement of important Phe7 in SP molecule by D-Phe7 a peptide with weak agonistic and some although weak antagonistic effect is obtained (Yamaguchi et al., 1979; Pernow, 1983). The present studies were undertaken in order to compare the effect in vitro of SP and some of its shorter analogues on the ATPases activity systems, which participate in active transport of ions in synaptosomal membranes of rat brain. We have tested shorter sequences than the undecapeptide, for instance the carboxy-terminal heptapeptide SP(5_~t)-(analogueA) by replacing Gin 5 with
Table 1. Primary structures of the four fragments of substance P analoguesstudied Analogues Abbreviated structure Primary structure A [< GIus,Tyrs]SPs-It < Glus, Gin,Phe, Tyrs, Leu,Met NH2 B [< Glue,Tyr8] SPs.ll < Glu6, Phe, Tyrs, Lcu,Met NH2 C [< Glu6,D-PheT]SPs-ii < Glue, D-Phe~, Phe,l.,¢u,Met NH2 D [ < Glue,D-Phes]SP~_ll < Glue, Phe, D-Phes, Leu,Met NH2 737
738
LILLA
LACHOWICZ
and
l ~
GRAZYNA
JANISZEWSKA
NrJK A T P o s e o c t i v i l ' , C o M g ATPosr~ o c ~ i v l t r
I S°
SOI0 ~00F ~00
oa c. o
o SP II Ii
i
z~
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i
h
II II
ta3
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o
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o
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o
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1oo
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II
r
r l
i
basal
cerebrat
hippocampuS
corte~
Fig. 1 o f SP(t u) a n d these a n a l o g u e s f r a g m e n t s o n the activities o f (Na, K ) a n d (Ca, M g ) A T P a s e s o f synaptosomal membranes obtained from cerebral cortex a n d h i p p o c a m p u s o f rat brain. MATERIALS AND METHODS Brains from adult male Wistar rats (200-250 g) were immediately divided on ice into two areas: cerebral cortex and hippocampus. The synaptosomal fractions from these areas were obtained according to the method of Booth and Clark (1978). The purity of synaptosomal fractions was monitored by electron microscopy and by enzyme markers (Lachowicz et al., 1983; Szkudlarek and Lachowicz, 1986). The synaptosomal membranes (SM) preparations were obtained after hypoosmotic shock o f synaptosomes (Lachowicz et al., 1984; Szkudlarek and Lachowicz, 1986). One part of SM preparations was incubated for 20 min at 30°C in standard medium pH 7.4 which contained 0.25 M sucrose and 10 m M Tris-HCl for the determination of the basic enzymes activities. The remaining quantity was incubated under the same conditions but with 10 # M o f SP (SchwarzMann) (mol. wt 1340) or four examinated analogues. After incubation, the applied agents were removed by washing and centrifugation (20 min at 100,000 g). The activity o f ATP-phosphohydrolase (EC 3.6.1.3) was determined by the assay in which formation o f A D P by ATPase was coupled with N A D H oxidation in the presence of excess pyruvate kinase, LDH and phosphoenolpyruvate (Schar-
schmidt et al., 1979). The formation of N A D + was measured spectrophotometrically at 340 nm. Total ATPase activity was measured in a reaction mixture containing 5 m M Mg 2+, 125mM Na ÷ and 12.5mM K ÷. (Na, K) ATPase activity (ouabain-sensitive) was obtained as the difference between total ATPase activity and Mg-ATPase activity which was measured in the presence o f 1 m M ouabain. (Ca, Mg) ATPase activity was determined in the presence of 0.1 mM Ca 2+, 5 m M Mg 2+ and 0.1 mM EGTA. Enzyme activities were expressed as #tool P d m g protein/hr. Protein of SM fraction was estimate by the method of Lowry et al. (1951). Each value is the average of 4 or 6 determinations. The results are expressed as means +_ SE in Table 2 and are presented in figures as percentage of basal activities. Statistical evaluation was done by Student's t-test; P < 0.05 was chosen as the level o f significance.
RESULTS
Our present results support the previously obtained d a t a ( L a c h o w i c z et al., 1983) t h a t u n d e c a p e p t i d e - - - S P m a r k e d l y s t i m u l a t e s t h e activities o f s n a p t o s o m a l m e m b r a n e s (Na, K ) a n d (Ca, M g ) A T P a s e in c e r e b r a l cortex and hippocampus. The administration of heptapeptide--analogue A, in w h i c h G l u 5 a n d P h % are s u b s t i t u t e d b y < Glu5 a n d Tyrs, respectively, c a u s e d a f u r t h e r increase o f (Ca, M g ) A T P a s e activity o f c e r e b r a l c o r t e x m e r e -
Table 2. The effects in vitro of SP and its shorter analogues on the activities of synaptosomal membranes (Na, K) and (Ca, Mg) ATPases. Values are expressed as per cent of basal activity Cerebral cortex (Na, K) ATPase (Ca, Mg) ATPase activity activity (%) (%) Basal activity Substance P 10gM Analogue A--10 gM heptapeptide (
Hippocampus (Na, K) ATPase (Ca, Mg) ATPase activity activity (%) (%)
100 (7.30 + 1.30) 200a _+ 12
100 (5.73 + 1.05) 186a + 6.0
100 (4.20 + 0.62) 300° _+30
100 (5.90 + 0.83) 540~ + 36
180° _+ 15
280a + 10
78 + 14
331° _+30
109 + 20
150° + 17
88 4-_10
230~ + 22
109 ___8.0
120 _+7.0
72a +_ 10
46~ _+5.0
97 + 19
95 __ 12
90 +__9.0
95 _+ 13
739
Peptides affect synaptosomal ATPase branes in comparison with the degree of activity after SP and against basal activity of enzymes. A significant increase of (Ca, Mg) ATPase activity in comparison with basal activity was found in hippocampal membranes but it was distinctly lower than after SP using. The (Na, K) ATPase activity of cerebral cortex after analogue A administration is similar to that induced by SP in hippocampus it does not alter from basal values. It may be concluded that the effect of analogue A is significantly lower than that of SP. The removal of one amino acid from heptapeptide together with substitution of Glu6 with < G l u 6 and Phe8 with Tyrs (analogue B) shows significant increase Of (Ca, Mg) ATPase activity in membranes of cerebral cortex and hippocampus in comparison with basal activity. We have shown that analogue B stimulates less effectively the (Ca, Mg) ATPase activity than SP in both examined areas. We also noted the lack of alteration in (Na, K) ATPase activity after analogue B in these areas. The replacement (analogue C) of the Gin 6 residue with < Glu6 and Phe 7 with D-Phe7 in hexapeptide did not produce any distinct differences in activities of the investigated enzymes from cerebral cortex; but, in hippocampus significant decreases of the enzymes activities were observed. When the analogue D was applied, less marked changes in activities of enzymes were found. The activity of both enzymes in cerebral and hippocampal SM were also not significantly different from basal activities. DISCUSSION The results of the present study have demonstrated that administration in vitro of SP induced more evident changes in (Ca, Mg) and (Na, K) ATPase activities in hippocampus than in cerebral cortex. The results of Inoue et al. (1984) suggest that when SP is released from nerve terminals, it is hydrolysed into ts-,)SP, which may play an important role in terminating the synaptic action of SP. It therefore seemed of great importance to study the activities of SP hepta- and hexapeptides-introducing substitutions in position 5, 6, 7 and 8 what conditioned agonistic and antagonistic features of the drug (Blumberg and Teichberg, 1981; Jones and Ople, 1982). The present data indicate that heptapeptide <~_~)SP ( < Glus, Tyrs) is more effective than SP in relation to (Ca, Mg) ATPase activity from cerebral cortex. In hippocampus, manifold increase of enzyme activity was observed but it did not equal that in the case of SP application. The removal of one amino acid residue from heptapeptide-(analogue B) ( < G l u 6, Tyr 8 ) caused a decrease of activation of (Ca, Mg) ATPase, and maintenance of (Na, K) ATPase activity on the basic values in both areas. This data suggest that reduction of one residue in the peptide chain and exchange of Gln6 to < Glu~ diminishes the activity peculitarity of tested analogue. As it was expected, the replacement of the Phe by the D----conformation in positions 7, and subsequently 8, did not induce an increase of activity of
the investigated enzymes, moreover analogue C ( < Glu6, D-PheT) significantly decreased the enzyme activities in hippocampus. It may be an argument for the fact that position 7 for biological activity of drug, is especially important and that the substitution of Phe in peptide chain with conformation D reduces the stimulatory effects of hexapeptide against enzymes responsible for the active cation transport in synaptosomal membranes of brain. Our observations are indirectly supported by the finding that substitution of L---conformation in some amino acids with D-conformation, practically determines the feature in these peptides of potential antagonists to tachykinins (Couture et al., 1985; Bailay et al., 1986; Featherstone et al., 1986). It is of much interest that (Ca, Mg) ATPase is more susceptible to the action of SP or some its analogues of fragments than (Na, K) ATPase. It concerns mainly the hippocampal membranes, which is not striking, because this area is known as very rich in SP-ergic receptors. At present, it is difficult to decide if the action of C-terminal fragments of SP and its analogues is governed by a common mechanism. On the other hand, the presented data suggests that some SP fragment analogues were found to be inactive, which supports the view of specific receptor--mediated action of SP (Reiser et al., 1982). Finally, one cannot ignore, that SP and its shorter analogue fragments may regulate the active cation transport in synaptosomal membranes in brain. It means that these processes as well as ion channels action appear to be the basis of mechanism of neuronal excitation induced by drugs from the family of tachykinins. Acknowledgement---Conducted under contract C.P.B.R. No
3.14 with the Polish Academy of Sciences. REFERENCES
Bailey S. J., Featherstone R. L., Jordan C. C. and Morton I. K. M. (1986) On examination of the pharmacology of two substance P antagonists and the evidence for tachykinin receptor. Br. J. Pharmac. 87, 79-85. Blumberg S. and Teichberg V. I. (1981) Activity of substance P analogs substituted and the Gly9 and Glu6 positions. Biochem. biophys. Res. Commun. 99, 752-758. Booth R. F. and Clark J. B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370. Couture R., Yashpal K., Henry J. L., Esther E. and Domenico Regoli (1985) Characterization of spinal actions of four substance P analogues. Eur. J. Pharmac. 110, 63-69. Deutch A. Y., Maggio J. E., Bannon M. J., Kalivas P. W., Tam S. Y., Goldstein M. and Roth R. H. (1985) Substance K and Substance P differentially modulate mesolimbic and mesocortical systems. Peptides 6, 113-122. Dun N. J. and Minota S. (1981) Effects of substance P on neurones of the inferior mesenteric ganglia of the guineapig. J. Physiol. (Lond.) 321, 259-271. Eardley D. and McGee R. (1985) Both substance P agonists and antagonists inhibit ion conductance through nicotinic acetylcholine receptors on PC 12 ceils. Eur. J. Pharmac. 114, 101-104. Featherstone R. L., Fosbracy P. and Morton I. K. M. (1986) A comparison of the effects of three substance P
740
LILLA LACHOWICZ and GRA:~'YNAJANISZEWSKA
antagonists on tachykinin-stimulated [3H]acetylcholine release in the guinea-pig ileum. Br. J. Pharmac. 87, 73-78. Inoue A., Nakata Y., Yajima H. and Segawa T. (1984) Active uptake system for substance P carboxyl-terminal heptapeptide (5-11) into a fraction from rabbit euriched in glial cells. Jap. J. Pharmac. 36, 137-145. Jones R. S. G. and Ople H. R. (1982) A structure-activity profile of substance P and some of its fragments on supraspinal neurones in the rat. Neurosci. Lett. 33, 67-71. Krynjevic K. (1977) Effects of substance P on central neurons in cats. In Substance P (Edited by von Euler U. S. and Pernow B.), pp. 217-230. Raven Press, New York. Lachowicz L., Janiszewska G. and Wojtkowiak R. (1984) Influence of Ca 2+ and substance P on the (Ca, Mg) ATPase (EC3.6.1.3) activity of synaptosomes from different areas of rat brain. Int. J. Biochem. 16, 337340. Lachowicz L., Janiszewska G., Wojtkowiak R. and Wojtkowiak Z. (1983) Ca2+Mg2÷-ATPase activity of synaptosome fraction and synaptosomal membranes from different areas of rat brain. Int. J. Biochem. 15, 163-165. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Minota S., Dun N. J. and Karczmar A. G. (1981) Substance
P-induced depolarization in sympathetic neurons: not simple K-inactivation. Brain Res. 216, 224-228. Nicoll R. A. (1978) The action of thyrotropin-releasing hormone, Substance P and related peptides on frog spinal motoneurons. J. Pharmac. exp. Ther. 207, 817-824. Pernow B. (1983) Substance P. Pharmac. Rev. 35, 119-123. Reiser G., Folkers K. and Hamprecht B. (1982) Substance P enhances cation permeability of neuronal cell lines. Regul. Peptides 5, 85-93. Scharschmidt B. F., Keeffe E. B., Blankenship N. H. and Ockner R. R. (1979) Kalidation of a recording spectrophotometric method for measurement of membrane-associated Mg- and Na, K-ATPase activity. J. Lab. Clin. Med. 93, 790-799. Szkudlarek J. and Lachowicz L. (1986) Effects in vitro of L-Glutamate and kainic acid on the ATPase activities of synaptosomal membranes from different areas of rat brain. Neurosci. Lett. 65, 304-3t0. Yamagucki I., Rackur G., Leban J. J., Bjorkroth U., Rossel S. and Folkers K. (1979) Synthesis and biological activity of analogs of substance P, modified for conformational information by D-amino acids. Acta chem. scand. B 33, 63~8. Ziegigiinsberger W. and Tulloch J. F. (1979) Effects of substance P on neurones in the dorsal horn of the spinal cord of the cat. Brain Res. 166, 273-282.
0020-71IX/87 $3.00+ 0.00 Copyright © 1987PergamonJournals Ltd
Printed in Great Britain. All rights reserved
A COMPARISON OF THE EFFECTS OF SUBSTANCE P A N D SHORTER A N A L O G U E S ON THE SYNAPTOSOMAL ATPases ACTIVITIES IN THE RAT BRAIN LILLA LACHOWICZand GRAZYNAJANISZEWSKA II Department of Biochemistry, Institute of Physiology and Biochemistry, School of Medicine, Lindleya 6, 90-131 L6d~, Poland (Received 24 November 1986)
Abstract--The influence in vitro of SP and C-terminal fragments of analogues SP(5_ll) (
INTRODUCTION The endogenous undecapeptide substance P (SP) is a member of a family of peptides known as the tachykinins which possess the conserved carboxyl terminal sequence-Phe-X--Gly-Leu-Met-NH2 (Deutch et al., 1985). More neuronal pathways of action have been demonstrated than for any of the other peptide transmitters. However, the mechanism of the excitatory action of SP on neuronal cells still remains unclear. Substance P has been reported to excite neurons by depolarizing them with a concomitant change in membrane resistance (Krnjevic, 1977; Nicoll, 1978; Zieglg/insberger and Tulloch, 1979). On the other hand, an increase in membrane resistance frequently accompanies substance Pinduced depolarization, an inactivation of the K + conductance, which has been suggested to be involved in the underlying mechanism. Further, a more complex mechanism has been proposed: a simultaneous increase in Na ÷ conductance and a decrease in K + conductance (Dun and Minota, 1981; Minota et al., 1981). It has become evident that some neuropeptides regulate the movement of ions down these concentration gradients, through ion channels in the membrane (Eardly and McGree, 1985). But distinctly less information concerns neuropeptide effects on active
ions transport, and therefore, on ATPases pumps, which are dependent on hydrolysis of ATP. Substitution of amino acid residues with D-amino acids is known to confer antagonistic activity. By replacement of important Phe7 in SP molecule by D-Phe7 a peptide with weak agonistic and some although weak antagonistic effect is obtained (Yamaguchi et al., 1979; Pernow, 1983). The present studies were undertaken in order to compare the effect in vitro of SP and some of its shorter analogues on the ATPases activity systems, which participate in active transport of ions in synaptosomal membranes of rat brain. We have tested shorter sequences than the undecapeptide, for instance the carboxy-terminal heptapeptide SP(5_~t)-(analogueA) by replacing Gin 5 with
Table 1. Primary structures of the four fragments of substance P analoguesstudied Analogues Abbreviated structure Primary structure A [< GIus,Tyrs]SPs-It < Glus, Gin,Phe, Tyrs, Leu,Met NH2 B [< Glue,Tyr8] SPs.ll < Glu6, Phe, Tyrs, Lcu,Met NH2 C [< Glu6,D-PheT]SPs-ii < Glue, D-Phe~, Phe,l.,¢u,Met NH2 D [ < Glue,D-Phes]SP~_ll < Glue, Phe, D-Phes, Leu,Met NH2 737
738
LILLA
LACHOWICZ
and
l ~
GRAZYNA
JANISZEWSKA
NrJK A T P o s e o c t i v i l ' , C o M g ATPosr~ o c ~ i v l t r
I S°
SOI0 ~00F ~00
oa c. o
o SP II Ii
i
z~
I
i
h
II II
ta3
t~
o
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o
•~
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h
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2OO
rt
tl
~1
i
I I
T,
Jl
f i t
1oo
Z
i r i r
II
r
r l
i
basal
cerebrat
hippocampuS
corte~
Fig. 1 o f SP(t u) a n d these a n a l o g u e s f r a g m e n t s o n the activities o f (Na, K ) a n d (Ca, M g ) A T P a s e s o f synaptosomal membranes obtained from cerebral cortex a n d h i p p o c a m p u s o f rat brain. MATERIALS AND METHODS Brains from adult male Wistar rats (200-250 g) were immediately divided on ice into two areas: cerebral cortex and hippocampus. The synaptosomal fractions from these areas were obtained according to the method of Booth and Clark (1978). The purity of synaptosomal fractions was monitored by electron microscopy and by enzyme markers (Lachowicz et al., 1983; Szkudlarek and Lachowicz, 1986). The synaptosomal membranes (SM) preparations were obtained after hypoosmotic shock o f synaptosomes (Lachowicz et al., 1984; Szkudlarek and Lachowicz, 1986). One part of SM preparations was incubated for 20 min at 30°C in standard medium pH 7.4 which contained 0.25 M sucrose and 10 m M Tris-HCl for the determination of the basic enzymes activities. The remaining quantity was incubated under the same conditions but with 10 # M o f SP (SchwarzMann) (mol. wt 1340) or four examinated analogues. After incubation, the applied agents were removed by washing and centrifugation (20 min at 100,000 g). The activity o f ATP-phosphohydrolase (EC 3.6.1.3) was determined by the assay in which formation o f A D P by ATPase was coupled with N A D H oxidation in the presence of excess pyruvate kinase, LDH and phosphoenolpyruvate (Schar-
schmidt et al., 1979). The formation of N A D + was measured spectrophotometrically at 340 nm. Total ATPase activity was measured in a reaction mixture containing 5 m M Mg 2+, 125mM Na ÷ and 12.5mM K ÷. (Na, K) ATPase activity (ouabain-sensitive) was obtained as the difference between total ATPase activity and Mg-ATPase activity which was measured in the presence o f 1 m M ouabain. (Ca, Mg) ATPase activity was determined in the presence of 0.1 mM Ca 2+, 5 m M Mg 2+ and 0.1 mM EGTA. Enzyme activities were expressed as #tool P d m g protein/hr. Protein of SM fraction was estimate by the method of Lowry et al. (1951). Each value is the average of 4 or 6 determinations. The results are expressed as means +_ SE in Table 2 and are presented in figures as percentage of basal activities. Statistical evaluation was done by Student's t-test; P < 0.05 was chosen as the level o f significance.
RESULTS
Our present results support the previously obtained d a t a ( L a c h o w i c z et al., 1983) t h a t u n d e c a p e p t i d e - - - S P m a r k e d l y s t i m u l a t e s t h e activities o f s n a p t o s o m a l m e m b r a n e s (Na, K ) a n d (Ca, M g ) A T P a s e in c e r e b r a l cortex and hippocampus. The administration of heptapeptide--analogue A, in w h i c h G l u 5 a n d P h % are s u b s t i t u t e d b y < Glu5 a n d Tyrs, respectively, c a u s e d a f u r t h e r increase o f (Ca, M g ) A T P a s e activity o f c e r e b r a l c o r t e x m e r e -
Table 2. The effects in vitro of SP and its shorter analogues on the activities of synaptosomal membranes (Na, K) and (Ca, Mg) ATPases. Values are expressed as per cent of basal activity Cerebral cortex (Na, K) ATPase (Ca, Mg) ATPase activity activity (%) (%) Basal activity Substance P 10gM Analogue A--10 gM heptapeptide (
Hippocampus (Na, K) ATPase (Ca, Mg) ATPase activity activity (%) (%)
100 (7.30 + 1.30) 200a _+ 12
100 (5.73 + 1.05) 186a + 6.0
100 (4.20 + 0.62) 300° _+30
100 (5.90 + 0.83) 540~ + 36
180° _+ 15
280a + 10
78 + 14
331° _+30
109 + 20
150° + 17
88 4-_10
230~ + 22
109 ___8.0
120 _+7.0
72a +_ 10
46~ _+5.0
97 + 19
95 __ 12
90 +__9.0
95 _+ 13
739
Peptides affect synaptosomal ATPase branes in comparison with the degree of activity after SP and against basal activity of enzymes. A significant increase of (Ca, Mg) ATPase activity in comparison with basal activity was found in hippocampal membranes but it was distinctly lower than after SP using. The (Na, K) ATPase activity of cerebral cortex after analogue A administration is similar to that induced by SP in hippocampus it does not alter from basal values. It may be concluded that the effect of analogue A is significantly lower than that of SP. The removal of one amino acid from heptapeptide together with substitution of Glu6 with < G l u 6 and Phe8 with Tyrs (analogue B) shows significant increase Of (Ca, Mg) ATPase activity in membranes of cerebral cortex and hippocampus in comparison with basal activity. We have shown that analogue B stimulates less effectively the (Ca, Mg) ATPase activity than SP in both examined areas. We also noted the lack of alteration in (Na, K) ATPase activity after analogue B in these areas. The replacement (analogue C) of the Gin 6 residue with < Glu6 and Phe 7 with D-Phe7 in hexapeptide did not produce any distinct differences in activities of the investigated enzymes from cerebral cortex; but, in hippocampus significant decreases of the enzymes activities were observed. When the analogue D was applied, less marked changes in activities of enzymes were found. The activity of both enzymes in cerebral and hippocampal SM were also not significantly different from basal activities. DISCUSSION The results of the present study have demonstrated that administration in vitro of SP induced more evident changes in (Ca, Mg) and (Na, K) ATPase activities in hippocampus than in cerebral cortex. The results of Inoue et al. (1984) suggest that when SP is released from nerve terminals, it is hydrolysed into ts-,)SP, which may play an important role in terminating the synaptic action of SP. It therefore seemed of great importance to study the activities of SP hepta- and hexapeptides-introducing substitutions in position 5, 6, 7 and 8 what conditioned agonistic and antagonistic features of the drug (Blumberg and Teichberg, 1981; Jones and Ople, 1982). The present data indicate that heptapeptide <~_~)SP ( < Glus, Tyrs) is more effective than SP in relation to (Ca, Mg) ATPase activity from cerebral cortex. In hippocampus, manifold increase of enzyme activity was observed but it did not equal that in the case of SP application. The removal of one amino acid residue from heptapeptide-(analogue B) ( < G l u 6, Tyr 8 ) caused a decrease of activation of (Ca, Mg) ATPase, and maintenance of (Na, K) ATPase activity on the basic values in both areas. This data suggest that reduction of one residue in the peptide chain and exchange of Gln6 to < Glu~ diminishes the activity peculitarity of tested analogue. As it was expected, the replacement of the Phe by the D----conformation in positions 7, and subsequently 8, did not induce an increase of activity of
the investigated enzymes, moreover analogue C ( < Glu6, D-PheT) significantly decreased the enzyme activities in hippocampus. It may be an argument for the fact that position 7 for biological activity of drug, is especially important and that the substitution of Phe in peptide chain with conformation D reduces the stimulatory effects of hexapeptide against enzymes responsible for the active cation transport in synaptosomal membranes of brain. Our observations are indirectly supported by the finding that substitution of L---conformation in some amino acids with D-conformation, practically determines the feature in these peptides of potential antagonists to tachykinins (Couture et al., 1985; Bailay et al., 1986; Featherstone et al., 1986). It is of much interest that (Ca, Mg) ATPase is more susceptible to the action of SP or some its analogues of fragments than (Na, K) ATPase. It concerns mainly the hippocampal membranes, which is not striking, because this area is known as very rich in SP-ergic receptors. At present, it is difficult to decide if the action of C-terminal fragments of SP and its analogues is governed by a common mechanism. On the other hand, the presented data suggests that some SP fragment analogues were found to be inactive, which supports the view of specific receptor--mediated action of SP (Reiser et al., 1982). Finally, one cannot ignore, that SP and its shorter analogue fragments may regulate the active cation transport in synaptosomal membranes in brain. It means that these processes as well as ion channels action appear to be the basis of mechanism of neuronal excitation induced by drugs from the family of tachykinins. Acknowledgement---Conducted under contract C.P.B.R. No
3.14 with the Polish Academy of Sciences. REFERENCES
Bailey S. J., Featherstone R. L., Jordan C. C. and Morton I. K. M. (1986) On examination of the pharmacology of two substance P antagonists and the evidence for tachykinin receptor. Br. J. Pharmac. 87, 79-85. Blumberg S. and Teichberg V. I. (1981) Activity of substance P analogs substituted and the Gly9 and Glu6 positions. Biochem. biophys. Res. Commun. 99, 752-758. Booth R. F. and Clark J. B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370. Couture R., Yashpal K., Henry J. L., Esther E. and Domenico Regoli (1985) Characterization of spinal actions of four substance P analogues. Eur. J. Pharmac. 110, 63-69. Deutch A. Y., Maggio J. E., Bannon M. J., Kalivas P. W., Tam S. Y., Goldstein M. and Roth R. H. (1985) Substance K and Substance P differentially modulate mesolimbic and mesocortical systems. Peptides 6, 113-122. Dun N. J. and Minota S. (1981) Effects of substance P on neurones of the inferior mesenteric ganglia of the guineapig. J. Physiol. (Lond.) 321, 259-271. Eardley D. and McGee R. (1985) Both substance P agonists and antagonists inhibit ion conductance through nicotinic acetylcholine receptors on PC 12 ceils. Eur. J. Pharmac. 114, 101-104. Featherstone R. L., Fosbracy P. and Morton I. K. M. (1986) A comparison of the effects of three substance P
740
LILLA LACHOWICZ and GRA:~'YNAJANISZEWSKA
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