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Heterogeneity of [125I]sarafotoxin S6b binding sites in rat tissues
1746 extracls were suspended in octyl-glucoside and fipid vesicles were prepared by dialysis in the presence of receptors. P h o s p ~ p i d s appeared to increase the insulin-stimulation of the tyrosine kinase activity and restored the enzymatic activity of the "'delipidate" receptor. Their efficiency was the following: brain phospholipids > placental phosphofipids + brain phosphatidyl > serine placental phospholipids + placental phosphatidylserine > placental phospholipids > soybean phc~holipids. Cholesterol added to the phospholipid vesicules had an inhibitory effect on the tyrosine kinase activation. The mechanism by which membrane lipids could modulate the coupling between insulin binding and tyrosine kinase activity is still obscure. However, it could be hypothesized that alterations in lipid composition may induce changes in the lateral mobility of lipids and proteins in the plasma membrane and thus affect the ability of the receptor to aggregate, segregate into specific membrane regions, a n d / o r exhibit optimal configuration for the subunit L ~ s i n e kinase activation after insulin binding.
References ikeneau, C. (1987) Biochim. Biophys. Acta 928, 287-296. Van Obberghen E. (1983) Proc. Natl. Acad. Sci. USA 80, 945-949.
P.th.0901
Heterogeneity of [ ,2s ||sarafotoxin S6b binding sites in rat tissues Jones, C.R. a n d Hiley, C.R. Merrell Dow Research Institute, B.P. 447 R / 9 , 16 rue d'Ankaro, 67009 Strasbourg and Dept. of Pharn.~'ologg, University of Cambridge, Tennis Court Road, Cambridge, U.K.
Sarafotoxin S6b is a cardiotoxic snake venom whose primary amino-acid sequence is highly homologous with the vasoconstrictor peptide endothelin (ET). It has been reported to bind to the same binding sites as ET in rat atrium and brain (Ambar et al., 1989). We therefore investigated [~ZSl]sarafotoxin S6b binding in rat aorta and cerebellum in order to evaluate this novel ligand and we have compared its binding in the cerebellum with that of [~25I]endothelin-1. Tissues were obtained from male Sprague-Dawley rats (200-300 g; Charles River France). Cerebella were homogenized in 10 vol. ice-cold buffer (50 mM HEPES; 1 mM 1,10 phenanthroline; pH 7.4) in a Teflon-in-glass homogenizer with 10 up-and-down strokes. Aortae, c u t / n t o 3 mm lengths, were homogenized in 7.5 vol. of the same buffer with 2 × 20s bursts in a Polytron at setting 7. Homogenates were centrifuged at 1000 g for 10 min at 4°C. Protein concentration in the supernatants, which were used for binding experiments, was determined by the Lowry method. Incubations (2 h at 25°C) were in 0.5 ml volume with 0.1-0.15 mg (cerebellum) or I mg (aorta) protein in a 50 mM HEPES buffer (pH 7.4) containing 1 mM 1,10 phenanthroline, 0.1~ bovine serum albumen and 1 4 0 / t g / m l bacitracin (all from Sigma Chemical Co). Bound and free ligand were separated by filtration through Whatman G F / B filters. Saturation experiments used 12 concentrations (7 pM-10 riM) of [lZSl]sarafotoxin S6b or [125I]ET-1 (Amersham International) and non-specific 0inding was defined by 1/tM ET-1. Inhibition experiments were identical except that a smg!e concentration (25-50 pM) of radioligand and 11 concentrations (1 pM-1 /tM) of ET-1 or sarafotoxin S6b (Peptide Institute, Osaka, Japan) were used. Specific [lZSl]sarafotoxin S6b binding was 71-91% of the total in the aorta and 91-98~ in the cerebellum. There were no differences in the displacement of this figand by either I/LM ET-1 or I / J M sarafotoxin S6b. In the cerebellum [lZSl]sarafotoxin S6b bound to a single binding site (Hill slope, h a , -- 0.90 ± 0.05; n -- 3) with a dissociation constant (K d) of 0.24 _+0.06 nM; maximum binding capacity was 233 ± 70 fmol/mg protein compared to 321 ± 58 fmol/mg protein ( n - - 5 ) for [lZSl]ET-1. In 3 aortic homogenates, analysis of [12Sl]sarafotoxin S6b binding isotherms with LIGAND (Munson and Rodbard, 1980) gave a significantly better fit to a 2-site model (overall n a -- 0.86 ± 0.05). The K d values were 3.0 _+ 1.5 nM and 145 ± 12 pM with relative abundances of 70 ± 18~ and 29 ± 17~ respectively. In inhibition studies in the cerebellum the K i values for sarafotoxin S6b inhibiting the binding of [12sl]sarafotoxin S6b and [lZSl]ET-1 were respectively 0.51 ± 0.07 nM (n n = 1.0 ± 0.1; n -- 3) and 0.23 ± 0.06 nM (n H ffi 1.1 ± 0.1; n ffi 6). The corresponding values for ET-1 were 0.64 ± 0.23 nM ([12SI]sarafotoxin S6b; n H = 0.9 ± 0.1; n -- 3) and 0.83 ± 0.15 nM ([lZSl]ET-1; n a -- 1.1 ± 0.1; n - 6).
1747 In conclusion [12Sl]sarafotoxin S6b is a useful tool for the study of ET-1 binding sites. In the cerebellum it appears to label the same population of sites as [1251]ET-1, has a very low non-specific binding and the interaction between sarafotoxin Stb and ET-1 appears to be competitive. In the aorta there appears to be more than one binding site for this cardiotoxin and the nature of these sites in relation to possible multiple receptors for members of the endothelin/sarafotoxin family of peptides remains to be determined. We are grateful to Amersham International for the generous gift of [12Sl]sarafotoxin S6b.
References Ambar, I., Kloog, Y., Schvartz, I., Hazum, E. and Sokolovsky, M., 1989, Biochcm. Biophys. Res. Commun. 158, 195. Munson, PJ. and Rodbard, D., 1980, Anal. Biochem. 107, 220.
I.th.09l Effects of putative intracellular calcium antagonists on striatal dopamine and acelylchoHne releases monitored in vivo with microdiaJysis Goiny, M., Guix, T. and Ungerstedt, U. Department of Pharmacology, Karolinska Instituter, S10401 Stockholm, Sweden
8-(N,N-Diethylamino)Octyl-3,4,5,-Trimethoxybenzoate (TMB-8), a putative intracellular calcium antagonist, has pronounced actions on pancreatic and adrenal secretions and on muscle contractions. In contrast, if intracerebroventricular injections of TMB-8 have been shown to produce antinociception (J. Pharm. Exp. Ther. 239, 320, 1986), a definite action on brain neurotransmission has not yet been reported. In this study we analysed, with the intracerebral microdialysis technique, the action of TMB-8 on striatal dopamine and acetylcholine release. Microdialysis probes, 2 mm long, (Carnegie Medic in AS, Stockholm) were implanted bilateraly in the dorsal part of the striatum of halothane-anaesthetized male Sprague-Dawley rats. The dialysis probes were perfused at a constant rate of 2 ul/min with Ringer solutions complemented with the drugs used. Dopamine and metabolites were assayed in 20 rain fractions on a reverse phase ion-pair high performance liquid chromatography (HPLC) system with electrochemical detection. Acetylcholine and choline were analysed using HPLC with post column enzymatic reactor and electrochemical detector (BAS, West Lafayette, In., U.S.A.) after inhibition of endogenous acetylcholinesterase with neostigmine. The inclusion of TMB-8 (10 -6 M-10 -3 M) in the Ringer solution produced an immediate, dose dependent, increase in perfusate dopamine levels within the first 20 rain period. Thereafter, dopamine rapidly fell under the initial levels. When the striatum, after perfusion with TMB-8 (10 -3 M) during 5 fractions, were further challenged with tyramine (10 -5 M) the dopamine release was less than 10~ of that in the control side. In contrast a challenge with 100 mM KCI produced a marked although delayed and prolonged increase in dopamine levels. Acetylcholine levels were elevated by the inclusion of TMB-8 in ~he perfusion medium. The effects of TMB-8 on the release of dopamine were mimicked by another putative intracellular calcium antagonist, dantrolene sodium. Pretreatment with the toxin tetrodotoxin, which abolishs nerve impulse, did not suppressed the stimulatory effect of TMB-8 (10-3 M) on the release of striatal dopamine. The results show that TMB-8 has also marked, although probably multiple, effects on brain neurotransmission. Considering the established dependency of striatal dopamine release upon calcium levels, the initial effect of TMB-8 would probably be mediated by an effect other than intracellular calcium antagonism, although the suppression of an inhibitory local modulation cannot actually be disregarded.
References ikeneau, C. (1987) Biochim. Biophys. Acta 928, 287-296. Van Obberghen E. (1983) Proc. Natl. Acad. Sci. USA 80, 945-949.
P.th.0901
Heterogeneity of [ ,2s ||sarafotoxin S6b binding sites in rat tissues Jones, C.R. a n d Hiley, C.R. Merrell Dow Research Institute, B.P. 447 R / 9 , 16 rue d'Ankaro, 67009 Strasbourg and Dept. of Pharn.~'ologg, University of Cambridge, Tennis Court Road, Cambridge, U.K.
Sarafotoxin S6b is a cardiotoxic snake venom whose primary amino-acid sequence is highly homologous with the vasoconstrictor peptide endothelin (ET). It has been reported to bind to the same binding sites as ET in rat atrium and brain (Ambar et al., 1989). We therefore investigated [~ZSl]sarafotoxin S6b binding in rat aorta and cerebellum in order to evaluate this novel ligand and we have compared its binding in the cerebellum with that of [~25I]endothelin-1. Tissues were obtained from male Sprague-Dawley rats (200-300 g; Charles River France). Cerebella were homogenized in 10 vol. ice-cold buffer (50 mM HEPES; 1 mM 1,10 phenanthroline; pH 7.4) in a Teflon-in-glass homogenizer with 10 up-and-down strokes. Aortae, c u t / n t o 3 mm lengths, were homogenized in 7.5 vol. of the same buffer with 2 × 20s bursts in a Polytron at setting 7. Homogenates were centrifuged at 1000 g for 10 min at 4°C. Protein concentration in the supernatants, which were used for binding experiments, was determined by the Lowry method. Incubations (2 h at 25°C) were in 0.5 ml volume with 0.1-0.15 mg (cerebellum) or I mg (aorta) protein in a 50 mM HEPES buffer (pH 7.4) containing 1 mM 1,10 phenanthroline, 0.1~ bovine serum albumen and 1 4 0 / t g / m l bacitracin (all from Sigma Chemical Co). Bound and free ligand were separated by filtration through Whatman G F / B filters. Saturation experiments used 12 concentrations (7 pM-10 riM) of [lZSl]sarafotoxin S6b or [125I]ET-1 (Amersham International) and non-specific 0inding was defined by 1/tM ET-1. Inhibition experiments were identical except that a smg!e concentration (25-50 pM) of radioligand and 11 concentrations (1 pM-1 /tM) of ET-1 or sarafotoxin S6b (Peptide Institute, Osaka, Japan) were used. Specific [lZSl]sarafotoxin S6b binding was 71-91% of the total in the aorta and 91-98~ in the cerebellum. There were no differences in the displacement of this figand by either I/LM ET-1 or I / J M sarafotoxin S6b. In the cerebellum [lZSl]sarafotoxin S6b bound to a single binding site (Hill slope, h a , -- 0.90 ± 0.05; n -- 3) with a dissociation constant (K d) of 0.24 _+0.06 nM; maximum binding capacity was 233 ± 70 fmol/mg protein compared to 321 ± 58 fmol/mg protein ( n - - 5 ) for [lZSl]ET-1. In 3 aortic homogenates, analysis of [12Sl]sarafotoxin S6b binding isotherms with LIGAND (Munson and Rodbard, 1980) gave a significantly better fit to a 2-site model (overall n a -- 0.86 ± 0.05). The K d values were 3.0 _+ 1.5 nM and 145 ± 12 pM with relative abundances of 70 ± 18~ and 29 ± 17~ respectively. In inhibition studies in the cerebellum the K i values for sarafotoxin S6b inhibiting the binding of [12sl]sarafotoxin S6b and [lZSl]ET-1 were respectively 0.51 ± 0.07 nM (n n = 1.0 ± 0.1; n -- 3) and 0.23 ± 0.06 nM (n H ffi 1.1 ± 0.1; n ffi 6). The corresponding values for ET-1 were 0.64 ± 0.23 nM ([12SI]sarafotoxin S6b; n H = 0.9 ± 0.1; n -- 3) and 0.83 ± 0.15 nM ([lZSl]ET-1; n a -- 1.1 ± 0.1; n - 6).
1747 In conclusion [12Sl]sarafotoxin S6b is a useful tool for the study of ET-1 binding sites. In the cerebellum it appears to label the same population of sites as [1251]ET-1, has a very low non-specific binding and the interaction between sarafotoxin Stb and ET-1 appears to be competitive. In the aorta there appears to be more than one binding site for this cardiotoxin and the nature of these sites in relation to possible multiple receptors for members of the endothelin/sarafotoxin family of peptides remains to be determined. We are grateful to Amersham International for the generous gift of [12Sl]sarafotoxin S6b.
References Ambar, I., Kloog, Y., Schvartz, I., Hazum, E. and Sokolovsky, M., 1989, Biochcm. Biophys. Res. Commun. 158, 195. Munson, PJ. and Rodbard, D., 1980, Anal. Biochem. 107, 220.
I.th.09l Effects of putative intracellular calcium antagonists on striatal dopamine and acelylchoHne releases monitored in vivo with microdiaJysis Goiny, M., Guix, T. and Ungerstedt, U. Department of Pharmacology, Karolinska Instituter, S10401 Stockholm, Sweden
8-(N,N-Diethylamino)Octyl-3,4,5,-Trimethoxybenzoate (TMB-8), a putative intracellular calcium antagonist, has pronounced actions on pancreatic and adrenal secretions and on muscle contractions. In contrast, if intracerebroventricular injections of TMB-8 have been shown to produce antinociception (J. Pharm. Exp. Ther. 239, 320, 1986), a definite action on brain neurotransmission has not yet been reported. In this study we analysed, with the intracerebral microdialysis technique, the action of TMB-8 on striatal dopamine and acetylcholine release. Microdialysis probes, 2 mm long, (Carnegie Medic in AS, Stockholm) were implanted bilateraly in the dorsal part of the striatum of halothane-anaesthetized male Sprague-Dawley rats. The dialysis probes were perfused at a constant rate of 2 ul/min with Ringer solutions complemented with the drugs used. Dopamine and metabolites were assayed in 20 rain fractions on a reverse phase ion-pair high performance liquid chromatography (HPLC) system with electrochemical detection. Acetylcholine and choline were analysed using HPLC with post column enzymatic reactor and electrochemical detector (BAS, West Lafayette, In., U.S.A.) after inhibition of endogenous acetylcholinesterase with neostigmine. The inclusion of TMB-8 (10 -6 M-10 -3 M) in the Ringer solution produced an immediate, dose dependent, increase in perfusate dopamine levels within the first 20 rain period. Thereafter, dopamine rapidly fell under the initial levels. When the striatum, after perfusion with TMB-8 (10 -3 M) during 5 fractions, were further challenged with tyramine (10 -5 M) the dopamine release was less than 10~ of that in the control side. In contrast a challenge with 100 mM KCI produced a marked although delayed and prolonged increase in dopamine levels. Acetylcholine levels were elevated by the inclusion of TMB-8 in ~he perfusion medium. The effects of TMB-8 on the release of dopamine were mimicked by another putative intracellular calcium antagonist, dantrolene sodium. Pretreatment with the toxin tetrodotoxin, which abolishs nerve impulse, did not suppressed the stimulatory effect of TMB-8 (10-3 M) on the release of striatal dopamine. The results show that TMB-8 has also marked, although probably multiple, effects on brain neurotransmission. Considering the established dependency of striatal dopamine release upon calcium levels, the initial effect of TMB-8 would probably be mediated by an effect other than intracellular calcium antagonism, although the suppression of an inhibitory local modulation cannot actually be disregarded.